a historical perspective on protein crystallization from 1840 to the present day

REVIEW ARTICLE

A historical perspective on protein crystallization from1840 to the present dayRichard Gieg�e

Institut de Biologie Mol�eculaire et Cellulaire, Universit�e de Strasourg et CNRS, France

Keywords

automation; crystal growth; high-throughput;

macromolecular assemblies; methods of

crystallization; microgravity; nucleation;

nucleic acids; protein crystallization; virus

Correspondence

R. Gieg�e, Architecture et R�eactivit�e de

l’ARN, Universit�e de Strasbourg, CNRS,

IBMC, 15 rue Ren�e Descartes, F-67084,

Strasbourg, France

Fax: +33 (0)3 88 60 22 18

Tel: +33 (0)3 88 41 70 58

E-mail: [email protected]

Note

The term ‘Protein’ is often taken as the

generic name for a biological

macromolecule or a macromolecular

assembly. ‘Protein solubility’ can have two

distinct meanings, either the amount of

protein that can be dissolved in a solvent or

the protein concentration in a solution in

equilibrium with a phase containing its

crystalline form. ‘Crystallants’ (often

referred as ‘precipitants’) and ‘nucleants’

are the chemical or physical factors that

promote nucleation and/or crystallization.

(Received 12 July 2013, revised 30 August

2013, accepted 27 September 2013)

doi:10.1111/febs.12580

Protein crystallization has been known since 1840 and can prove to be

straightforward but, in most cases, it constitutes a real bottleneck. This stim-

ulated the birth of the biocrystallogenesis field with both ‘practical’ and

‘basic’ science aims. In the early years of biochemistry, crystallization was a

tool for the preparation of biological substances. Today, biocrystallogenesis

aims to provide efficient methods for crystal fabrication and a means to opti-

mize crystal quality for X-ray crystallography. The historical development of

crystallization methods for structural biology occurred first in conjunction

with that of biochemical and genetic methods for macromolecule production,

then with the development of structure determination methodologies and,

recently, with routine access to synchrotron X-ray sources. Previously, the

identification of conditions that sustain crystal growth occurred mostly

empirically but, in recent decades, this has moved progressively towards

more rationality as a result of a deeper understanding of the physical chemis-

try of protein crystal growth and the use of idea-driven screening and high-

throughput procedures. Protein and nucleic acid engineering procedures to

facilitate crystallization, as well as crystallization methods in gelled-media or

by counter-diffusion, represent recent important achievements, although the

underlying concepts are old. The new nanotechnologies have brought a sig-

nificant improvement in the practice of protein crystallization. Today, the

increasing number of crystal structures deposited in the Protein Data Bank

could mean that crystallization is no longer a bottleneck. This is not the case,

however, because structural biology projects always become more challeng-

ing and thereby require adapted methods to enable the growth of the appro-

priate crystals, notably macromolecular assemblages.

Introduction

The art of crystallization dates back to antiquity and,

for a long time, primarily comprised the growth of salt

crystals by evaporation procedures. Protein crystalliza-

tion is much more recent and appeared in the first half

Abbreviations

2D, two-dimensional; aaRS, aminoacyl-tRNA synthetase (e.g. AspRS for aspartyl-tRNA synthetase, PheRS for phenylalanyl-tRNA synthetase,

etc.); AFM, atomic force microscopy; DLS, dynamic light scattering; HEW, hen egg-white; ICCBM, International Conference on the

Crystallization of Biological Macromolecules; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; PDB, Protein Data Bank;

SANS, small-angle neutron scattering; SAXS, small-angle X-ray scattering; TEW, turkey egg-white; TMV, tobacco mosaic virus.

6456 FEBS Journal 280 (2013) 6456–6497 ª 2013 FEBS

of 19th Century, with an initial publication in 1840 on

the observation of crystallites in blood preparations [1],

which in fact were haemoglobin crystals. Over the years,

the diversity of crystallized proteins has expanded,

although crystallization often occurred by chance and

using empirical procedures. For approximately one cen-

tury, crystallization was used as a means of protein

purification and characterization by biochemists and

physiologists. The situation changed when X-ray crys-

tallography entered biology in 1934 after the first X-ray

photograph of a protein crystal was taken [2]. Improve-

ments in crystallization procedures and the fabrication

of crystals suitable for structure determination arose in

parallel with advances in X-ray crystallographic meth-

ods and the ambition of structural biologists who were

seeking to image the macromolecular components of

living organisms. This became possible as a result of

interdisciplinary efforts merging biochemistry/molecu-

lar biology, chemistry, physics and engineering, which

gradually transformed the field of protein crystallization

into a scientific discipline of its own. I have named this

discipline ‘crystallogenesis’ [3], where the aim is to

understand and control crystal growth and quality; note

that a German version, ‘Krystallogenese’, was already

proposed in the 19th Century by different individuals,

such as Preyer [4]. The literature on biocrystallogenesis

is manifold. The present review restricts itself to a few

introductory references on historical [3,5–7] as well as

on methodological and physicochemical [8–16] aspectsand to a selection of most significant research articles

and focused reviews. More citations on facts listed in

Tables and Figures are provided in Data S1 to S10.

Additional bibliographic sources, particularly books,

reviews and International Conference on the Crystalli-

zation of Biological Macromolecules (ICCBM) Pro-

ceedings, are given in Data S11.

The present review is divided into three sections

describing how biocrystallogenesis emerged and became

a mature field, as well as how it became seminal for mod-

ern structural biology. They cover: (a) the period of phys-

iological and colloidal chemistry before the birth of

protein X-ray crystallography; (b) the early years of

structural biology when conventional methods of protein

crystallization were established; and (c) the years of more

recent technologies and structural genomics. The conclu-

sion outlines perspectives and sketches a few applications

beyond the field of structural biology (e.g. in medicine).

The time of physiology and chemistry(1840–1934)

In the 19th and early 20th Centuries, knowledge on

proteins was elusive and the name ‘protein’ (coined by

Berzelius in 1838) was not of universal use in biology

and chemistry. Terms such as ‘Proteid/Eiweissk€orper’

substances, ‘albumineous’ material or ‘colloids’ were

often employed for these mysterious substances. How-

ever, during this period, a few visionary physiologists,

chemists and physico-chemists established the corner-

stones of modern biology, notably structural biology,

when they worked out protocols leading to the pro-

duction of crystalline proteins. The basic methods of

protein crystallization were established and the essen-

tial physico-chemical properties of proteins discovered.

Crystallinity of haemoglobin and plant globulins

In 1840, Friedrich Ludwig H€unefeld published a book

entitled Der Chemismus in der thierischen Organisation

(Chemical Properties in the Animal Organization) in

which he reported (p. 160 and 161) how he acciden-

tally discovered the formation of crystalline material in

samples of earthworm blood held under two glass

slides and occasionally observed small plate-like crys-

tals in desiccated swine or human blood samples [1].

These were crystals of ‘haemoglobin’, a name coined

1864 by Felix Hoppe-Seyler for the ‘colorant substance

of blood’ [17]. In the following years, and likely even

before H€unefeld, many scientists observed haemo-

globin crystals when examining various animal tissues

or animal blood (e.g. Julius Budge, Otto Funke,

Albert von K€olliker, Karl G Lehmann, Franz Leydig

and Karl Reichert) but except Funke did not investi-

gate further the properties of these crystals [4].

In 1855, Theodor Hartig discovered a second family

of crystalline proteids in the gluten flour ‘Klebermehl’

from the Bertholletia excelsa Brazil nut [18]. Soon,

‘crystalloids’, as they were named, of globulins were

described by several authors in extracts of other plant

seeds (e.g. from Avena, Camelia, Crocus, Croton and

Ricinus), notably by Heinrich Ritthausen [19] and

mostly by Thomas B. Osborne [20] who knew and

extended Ritthausen’s work. By 1889, when Osborne

started his thorough biochemical work on plant globu-

lins, his main interest was to prepare pure specimens

of globulins by employing all of the available methods

at the time (particularly crystallization) to ensure

homogeneity of the preparations. As a result, he

obtained crystals of several globulins (two examples

are provided in Fig. 1A) and assigned them specific

designations; for example, ‘excelsin’ for the globulin

from Brazil nut (an allergen presently known as the

Ber e 2 protein) [21] and ‘edestin’ or ‘avenalin’ for

those from hemp seeds or oat kernels [20]. In 1907,

Osborne published a monograph summarizing his

investigations (revised in 1924) in which he described

FEBS Journal 280 (2013) 6456–6497 ª 2013 FEBS 6457

R. Gieg�e Protein crystallization for structural biology

procedures to obtain crystals that are based, amongst

others, on protein extractions from warm salt solutions

(40–60 °C) followed by slow cooling to room tempera-

ture; for further details, see the online version of the

original 1924 publication [22].

Deliberate protein crystallizations in the 19th and

early 20th Centuries

After the seminal findings by H€unefeld and Hartig,

many other physiological chemists and botanists tried

to deliberately produce crystals of haemoglobin and

plant globulins using more controlled protocols [6].

Thus, in 1851, Funke described how to grow human

haemoglobin crystals by successively diluting red blood

cells with solvents such as pure water, alcohol or ether,

followed by slow evaporation of the solvent from the

protein solution [23]. This was the first use of organic

solvents in protein crystallization. In 1871, the English-

born physiologist William T. Preyer, Professor at Uni-

versity of Jena, published a book entitled Die Blutk-

rystalle (The Crystals of Blood), reviewing the features

of haemoglobin crystals from ~ 50 species of mammals,

birds, reptiles and fishes [4]. Franz Hofmeister entered

the theater of crystal science in 1890 when he crystal-

lized hen egg-white (HEW) albumin [24].

The interest in haemoglobin crystals did not decline

in the 20th Century and was first highlighted in 1909

when the physiologist Edward T. Reichert, together

with the mineralogist Amos P. Brown, published an

impressive treatise on the preparation, physiology and

geometrical characterization of haemoglobin crystals

from several hundreds animals, including extinct

species such as the Tasmanian wolf (Thylacyanus cy-

nocephalus) [25] (Fig. 1B,C). The crystallization of

other proteins was also actively pursued in the first

half of the 20th Century. As was common practice in

chemistry, crystallization became a powerful step in

purification protocols. Examples are the crystalliza-

tion of animal and plant globulins (e.g. various serum

albumins and canavalin from jack beans), the crystal-

lization of a plant lectin (concanavalin A), the crys-

A

B

C

Fig. 1. Animal haemoglobins and plant

globulins, comprising the first animal and

vegetable proteins that were crystallized.

(A) Crystals of B. excelsa exelsin from the

Brazil nut (left) and of Avena sativa

avenalin from oat kernels (right) [20]. (B,

C) Haemoglobin crystals of the Tasmanian

wolf: (B) photographs of a-oxyhaemoglobin

showing groups of plates in parallel

growth (left) and of b-oxyhaemoglobin

showing small dodecahedral crystals

(right); (C) schematized drawing of the

above crystals emphasizing their prismatic

(left) and dodecahedral (right) habits [25] .


Protein crystallization for structural biology R. Gieg�e

tallization of several enzymes (carboxypeptidase, cata-

lase, chymotrypsin, ribonuclease, pepsin, trypsin, ure-

ase, etc.), the crystallization of the diphtheria toxin,

and the crystallization of the polypetidic hormone

insulin [6]. All of the investigators noted the impor-

tance of salts, organic solvents, pH and/or tempera-

ture for crystallization. Progressively, they took

advantage of the new ideas of Hofmeister on salt

effects (especially the ‘salting in’ and ‘salting out’

phenomena) to reach supersaturation and discovered

the crucial role of metal ions in protein crystalliza-

tion, notably for insulin crystallization where Zn2+

ions are indispensable [26].

Precursors that impacted upon the field of

protein crystallization

The biographical notes of pioneers and the highlights

of their achievements in crystal science are summarized

in Table 1. Besides H€unefeld, Funke and Hartig who

opened the field, the inspired physico-chemical contri-

butions of Hofmeister and Ostwald deserve particular

attention, although they were of indirect influence on

early crystallization investigations. Hofmeister was the

first individual to systematically study the effects of

salts on protein stability and solubility [27]. He is the

father of what is presently known as the Hofmeister

lyotropic salt series, which ranks the relative influence

of ions on the physical behaviour of proteins [28].

These salt effects (with NH4+ having the strongest

effect with respect to decreasing solubility) turned out

to be critical for understanding protein crystallization

[29,30]. Ostwald established the rules for time depen-

dent phase changes in chemical mixtures (solid–liquidtransitions) and discovered the phenomenon of ripen-

ing [31] that has found recent applications in macro-

molecular crystallization [32].

Reichert and Brown aimed to correlate the classifi-

cation of animal species with their evolution on the

basis of the morphology of their haemoglobin crystals

[25]. Today, this appears naive but, by 1909, the idea

underlying their work was in some way visionary

because, in present biology, evolution is accounted for

by protein sequences and three-dimensional structures.

In a more crystallographic perspective, they were the

first individuals to thoroughly describe polymorphism

in protein crystals, which is now amply demonstrated.

The motivation of James B. Sumner was different.

In 1917, when he was at Cornell University and had

heavy teaching obligations, he decided to accomplish

something of real importance during his spare time.

This was the risky project of purifying an enzyme.

Fortunately, using urease from the jack bean, he opted

for a good experimental model. Two years later, he

obtained crystals of the lectin concanavalin, which is

abundant in the jack bean. It took him an additional

7 years to find the appropriate recipe to prepare crys-

talline urease. The clue to success was the extraction

of the enzyme from the protein bulk with 30% alcohol

[33]. However, his 1926 paper, in which he reported

that solutions of dissolved urease crystals possess ‘to

an extraordinary degree the ability to decompose urea

into ammonium carbonate’ [33] generated skepticism

and his conclusion was rejected by the renowned Ger-

man organic chemist Willst€atter (1915 Nobel Prize in

Chemistry), who was convinced that the catalytic

activity of enzymes is a result of organic compounds

copurified or adsorbed on carrier proteins [5]. This

forced Sumner to provide stronger arguments and

stimulated John H. Northrop to study the crystalliza-

tion of swine pepsin for which he had strong biochem-

ical evidence of its protein nature [34]. Despite

intensive efforts, no putative catalytic entity could be

separated from either urease or pepsin. The contro-

versy was resolved when Northrop developed better

quantitative tools to purify, characterize and crystallize

proteins, and thereby generalized the concept of cata-

lytic proteins to pepsin, trypsin and chymotrypsin [35].

By 1937, Sumner closed the debate with a decisive

publication on catalase from beef liver showing that its

catalytic activity requires both the protein and an iron

porphyrin group [36]. Both Sumner and Northrop

received the Nobel Prize in Chemistry in 1946 for these

biochemistry-focused contributions [5]. They shared

the Nobel Prize with Wendel M. Stanley, who was the

first to have prepared a crystalline virus [37], although

he did not immediately realize the implications of his

finding as he was on a quest to prepare protein con-

stituents of tobacco mosaic virus (TMV) [38].

More influential from the viewpoint of crystal science

was Arda A. Green with her seminal papers on the phys-

ical chemistry of proteins completed in a continuation of

the early observations of Hofmeister on the solubility of

horse carboxy- and oxyhaemoglobin as a function of the

concentration of various salts, pH and temperature

[39,40]. Accordingly, she deduced an empirical relation-

ship between protein solubility and ionic strength

log S = b – Ksl

where S is solubility and l is ionic strength, Ks is the

salting-out constant considered to be independent of

pH and temeperature, and b is a protein-, pH- and

temperature-dependent constant). Interestingly, she

noted decreasing values of Ks correlated with the rank-

ing of the salts in the Hofmeister series. Arda

A. Green was active in many other domains of protein



Table 1. Early pioneers that impacted upon the emerging science of crystallogenesis. For references, see text and Data S1.

Pioneers Highlights Biographical notes

Friedrich L.

H€unefeld

1840: First observation of crystals in blood

samples (haemoglobin)

German MD and chemist, b.1799 M€uncheberg – †1882

Greifswald. Was active at Greifswald University (Professor of

Chemistry and Mineralogy). Was with Berzelius in 1827

Otto Funke 1851: First deliberate crystallization of

haemoglobin (Blutfarbstoffes) by evaporation

German MD and chemist, b.1828 Chemnitz – †1879 Freiburg.

Was active at Leipzig, then Freiburg University (Professor of

Medicine, then Physiological Chemistry)

Theodor Hartig 1855: Crystalline particles from extracts of the

Brazil nut (a storage protein known as

B. excelsa excelsin)

German forestry biologist and botanist, b.1805 Dillenburg – †1880

Brauschweig. Was active at the German Forestry Organization

Franz Hofmeister 1888: Different salts can be placed in a regular

order with respect to their salting-out effect

on proteins (ranking now known as the

‘Hofmeister series’ or ‘lyotropic series’)

1890: First crystals of HEW albumin

Bohemian-German MD, physiologist, chemist and pharmacologist,

b.1850 Prague – †1922 W€urzburg. Worked at Prague University

until 1896 (Professor of Pharmacology), succeeded Hoppe-Seyler

1896 at Strasbourg University, left for W€urzburg in 1919

Wilhelm Ostwalda 1897: Phenomenon of ripening describing the

change of an inhomogeneous structure over

time (called Ostwald ripening). Applies to

proteins, where large crystals can grow at the

expense of small ones

Baltic-German physical chemist and philosopher, b.1853 Riga –

†1932 Grossbothen. Educated in Tartu; worked at Riga

(1881–87) then Leipzig University (Professor of Chemistry and

Philosophy). Nobel Prize in Chemistry in 1909

Edward T. Reichert

and

Amos P. Brown

1909: Publication of an impressive opus on the

solubility, crystallization and crystal

characterization (shape, angles, etc.) of

haemoglobins from ~ 100 mammalian species

and a few Batrachia, birds, fishes and reptiles

American MD from the Medical Department of Pennsylviana

University, b.1855 – †1931. Educated in Berlin, Leipzig and

Geneva; worked mainly at University of Pennsylvania (Professor

of Physiology)

American mineralogist, b.1864 Germantown – †1918 Philadelphia.

Was head Professor of Department of Mineralogy and Geology,

University of Pennsylvania, Philadelphia, PA

James B. Sumner 1919: Crystals of Canavalis ensiformis

concanavalin A & B (jack bean)

1926: First crystallization of an enzyme, urease

from jack bean. Despite skepticism he claimed

that the crystalline enzyme is a protein

American chemist and biochemist, b.1887 Canton, MA – †1955

Buffalo, NY. Graduated from Harvard University; most research

at Cornell University, Ithaca, NY (Professor of Biochemistry).

Nobel Prize in Chemistry in 1946

John H. Northrop 1930: Pepsin in crystalline form. Northrop was

visionary in realizing that a crystalline form of a

protein is not in itself a criterion of purity

American biochemist, b.1891 Yonkers, NY – †1987

Wickenburg, AZ. Main work at Rockfeller Institute in New York,

NY, and Princeton, NJ. Nobel Prize in Chemistry in 1946

1931–33: Crystallization of trypsin and

chymotrypsin

Wendel M. Stanley 1930–40: Use of chemical methods, including

crystallization, for isolation of active substances

from viruses that are harmful to plants. In 1935,

isolated tobacco mosaic virus in crystalline form

American biochemist and virologist, b.1891 Ridgeville, IN – †1987

Salamanca, Spain. Main work at the Rockfeller Institute in

Princeton, NJ; after 1948 at University of California, Berkeley, CA

(Professor of Biochemistry). Nobel Prize in Chemistry in 1946

Arda A. Green 1931–32: Seminal papers on the solubility of

horse haemoglobin as a function of pH, ionic

strength and temperature

American protein chemist and biochemist, b.1899 Prospect, PA –

†1958 Baltimore. Many prominent scientists worked under

A. Green (e.g. Krebs, 1992 Nobel Prize) or were associated with

her (e.g. the Cori’s, 1947 Nobel Prize). Posthumous Garvan

Medal awarded to notable women chemists

1956: Crystallization of luciferase (her last

contribution)

John D. Bernal

and

1934: First X-ray diffraction pattern of a protein

crystal (pepsin)

British crystallographer, b.1901 Nenagh, Ireland – †1971 London.

Mentor of D. Hodgkin at Cambridge University (Professor of

Physics); 1937: moved to Birkbeck College, London (Professor of

Crystallography)

Dorothy (Crowfoot)

Hodgkin

British chemist and protein crystallographer, b.1910 Cairo – †1994

Ilmington. Educated in Oxford; was in Cambridge with J. Bernal

and held a post at Sommerville College, Oxford, until 1977.

Nobel Prize in Chemistry in 1964

aHis son Wolfgang Ostwald (1883–1943) was the initiator of colloid chemistry and biochemistry.



science while working with the most famous American

biochemists and this explains why her work on protein

solubility did not receive the recognition that it

deserves, although it did influence the two Cori’s and

Krebs, and all three were awarded a Nobel Prize, and

later impacted decisively upon the whole field of pro-

tein crystallization.

Considerations on protein crystallization in the

epoch of physiology and chemistry

In this epoch, crystallization was a tool for protein puri-

fication and was instrumental to demonstrate that the

catalytic activity of enzyme resides within the protein

itself. In the 19th Century, most crystalline proteins

were of plant origin and only a few animal proteins

(haemoglobins and albumins) were characterized as

pure substances. Crystals were obtained in the micro-

scale range by desiccation/evaporation procedures of

crude biological materials, mainly from extracts treated

by water, alcohol, hot acetic acid or salts to solubilize

their ‘albumineous’ entities. Scaling-up procedures rep-

resented a challenge that was first tackled by Preyer

with haemoglobin [4] and pursued by the biochemists in

the early 20th Century, who significantly enriched the

repertoire of crystalline proteins. All of these proteins

were easily available and had rather robust structures, a

feature not known at the time. In retrospect, one can

wonder why the early investigators were not intrigued

by the fact that proteins considered as colloidal sub-

stances with an elusive structure can be crystallized.

Being physiologists and biochemists, it is fortunate that

they were not refrained by the rules of classical crystal-

lography, which claim that crystals are formed by

strictly identical entities, although, today, it is well

established that macromolecular crystals can encompass

proteins with disordered domains.

The paradigm change in the field occurred in 1934

when John Desmond Bernal and Dorothy Crowfoot

(Hodgkin), two prominent figures in British science,

reported the first diffraction pattern of a protein crys-

tal [2]. This closed the epoch of chemistry and physiol-

ogy in biocrystallogenesis and marked the beginning of

structural biology.

The birth of biocrystallogenesis as ascience (1934–1990)

Growing crystals was not the major concern for the

pioneers of structural biology who were busy establish-

ing methods for structure determination. They used

proteins available in large amounts and easy to crystal-

lize with the bulk methods worked out by the biochem-

ists (see above). Once the first protein structures were

solved in the 1950/60s (Table 2), researchers became

more ambitious and enrolled in objective-focused pro-

Table 2. Landmarks inmacromolecule crystallization leading to three-

dimensional (3D) structures. For references, see text andData S2

Macromolecular

class

Subclass

Example of

crystallized entities

Name, origin, (year)a

3D structure

(year)a and

PDB codeb

Proteins

Globins Haemoglobin,

human (1840)

(1963) – 4hhb

Phytoglobulins Excelsin, Brazil nut

(1855)

(2007) – 2lvfc

Enzymes HEW lysozyme (1890) (1965) – 1lyz

Urease, jack been (1926) (2012) –4h9m

Pepsin, pig (1929) (1990) – 1pep

Catalase, beef liver (1937) (1985) – 7cat

Hormones Insulin, rabbit (1926) (1969) – 4insd

Toxins Erabutoxin, sea snake (1971) (1989) – 5ebx

Antibodies Intact IgG, human (1969) (1973) – 7fab

Membrane

proteins

Porin, Escherichia coli (1980) (1995) – 1opf

Sweet tasting

proteins

Thaumatin, Thaumatococcus

daniellii (1975)

(2002) – 1kwne

Nucleic acids

tRNAs tRNAPhe, Saccharomyces

cerevisiae (1968)

(1974) – 1tn2

DNA fragments Synthetic DNA

duplexes (1988)

(1989) – 2d13f

Supramolecular

assemblies

Viruses Plant virus, TMV (1935) (1986) – 1vtmg

Enzyme:RNA

complexes

AspRS:tRNAAsp,

S. cerevisiae (1980)

(1991) – 1asy

Membrane

embedded

assemblies

Photosynthetic

reaction center

Rhodopseudomonas

viridis (1982)

(1986) – 1prc

Protein:DNA

complexes

Nucleosome,

Xenopus laevis (1984)

(1998) – 1aoi

Ribosomes 70S,

Bacillus stearothermophilus

(1980)

(2001) – 1giyh

a Prime publication(s) of crystallization or structure.b PBD codes can correspond to a refined structure deposited after

the prime publication.c Crystal structure not yet in PDB, although NMR structure has

been solved.d PBD codes can also correspond to a structure of different taxo-

nomic origin than the first crystallized entity.e Structure at 1.2 �A resolution solved from crystals grown under

microgravity in gel with data collected at room temperature.f Example of a structure of an A-DNA decamer at 2 �A resolution.g Fibre-diffraction structure at 3.5 �A resolution.h First X-ray structure of a ribosome (i.e. that of Thermus thermo-

philus) at moderate resolution (5.5 �A).



jects. The supply of interesting proteins became a limit-

ing factor and the fabrication of crystals turned out to

be a major bottleneck. Studies aiming to understand

the functioning of enzymes and the mechanisms of

protein synthesis were probably the first emblematic

objective-focused projects that stimulated worldwide

interdisciplinary efforts to overcome this bottleneck

(e.g. for understanding tRNA biology) [7]). Further-

more, deciphering protein synthesis enlarged the prob-

lem of protein crystallization to nucleic acids [41] and

nucleoprotein complexes [42]. Crystallization of mem-

brane proteins was the other great challenge [43].

Widening and exploring the crystallization

parameter space

Crystallization processes are multiparametric phenom-

ena and therefore the primordial duty of experimenters

is to properly choose the parameters leading to best

crystal growth. In the field of protein crystallization,

early investigators were not always aware of this fact

and often obtained crystals by chance, although it was

soon noted that some factors were of importance, such

as the solubility of the protein, the type of salts used to

induce supersaturation, the temperature, the need for

metal ions, and the source and amount of the protein.

Nevertheless, many crystallographers considered pro-

tein crystallization as an art where magic skills are essen-

tial for success. This idea remained popular for some

time, especially because the amount of material avail-

able for crystallization purposes was often limited. This

prevented systematic studies aiming to understand the

global or specific effects brought by the known parame-

ters affecting protein crystallization [10] (Table 3).

When projects became more ambitious, the poor suc-

cess rate in crystallization attempts led a few pioneers

to develop methods better adapted to the requirements

of nascent structural biology. The aim was to produce

the rather large crystals needed at the time for diffrac-

tion measurements with limited amounts of protein

material [44] and, importantly, to enable an exploration

of the huge crystallization parameter space (Table 3).

Handling the diversity of parameters then became

another motivation to devise new crystallization proce-

dures. Thus, in the 1980s, ~ 90 different crystallants

were tested, with ammonium sulfate and poly(ethylene

glycol) 6000 ranking at the first places [9].

From conventional and forgotten methods to

project-driven approaches

Batch and dialysis methods were commonly employed

to obtain protein crystals for X-ray crystallography

(Table 4). In conventional batch methods, supersatu-

rated protein solutions containing all the required

ingredients are left undisturbed in sealed vessels. How-

ever, the success of crystallization, notably in terms of

number and size of grown crystals, is dependent on

the level of supersaturation at time zero, which should

be chosen and tuned appropriately. Accordingly, con-

ditions can easily be varied by temperature changes or

the addition of small aliquots of chemicals in the

experimental vessels. Alternatively, sealed crystalliza-

tion chambers can be opened to allow concentration

changes by evaporation. An attractive variation of the

conventional batch method is a sequential extraction

procedure by ammonium sulfate, which applies tem-

perature gradients on protein solutions at high ionic

strength [45]. It was validated with several proteins

and employed for crystallizing E. coli MetRS [46].

Similarly, in dialysis methods, modification or

exchange of the solutions in which the dialysis bags

are immersed allows tuning of the experimental condi-

tions. However, the main drawback of both methods

is the large volume of samples (in the millilitre range)

and, consequently, the large amounts of material

(> 10 mg) required for each assay.

The advent of molecular biology and the first suc-

cesses of X-ray crystallography stimulated biologists

and crystallographers to embark on ambitious pro-

jects. This was a driving force to devise adapted crys-

tallization methods. A initial breakthrough with an

immediate impact on structural biology came in 1968

with the invention of user-friendly vapour-diffusion

methods; first, in a sitting drop version for the crystal-

lization of tRNAs [47] that rapidly evolved in a num-

ber of variants, notably hanging and sandwiched

drops displayed in various experimental arrangements.

The method is based on the equilibration of a drop

with the protein to be crystallized and all ingredients

for crystallization against a reservoir containing the

crystallant at a higher concentration than in the drop.

Equilibration proceeds by diffusion of the volatile spe-

cies (e.g. water in most cases, although it can be

organic solvents or ammonia always present in ammo-

nium sulfate) until the vapour pressure of the drop

equals that of the reservoir, which is accompanied by

a volume decrease in the drop and an increase of the

protein concentration that can enter in the supersatu-

rated phase during which crystallization can occur.

The method can operate in a reverse regime if the ini-

tial concentration of the crystallant in the reservoir is

lower than that in the drop. In that case, water

exchange occurs from the reservoir to the drop. The

reverse vapour-diffusion method was discovered fortu-

itously in the course of an attempt to gently dissolve a



Table 3. Parameters affecting protein crystallization. For references, see text and Data S3.

Main parametersa Commentsa

Chemical and biochemical

Macromolecule Can be considered as the most important parameter

Purity Purity and homogeneity essential but not absolute prerequisites

Concentration Mostly in the range 5–20 mg�mL�1 (but examples at 1 mg�mL�1 or > 60 mg�mL�1)

Deliberate modification Chemical modification of amino acids, fragmentation into structural domains

Crystallants > 40 Single compounds and ~ 40 associations of two or more compounds

Saltsb 22 Compounds, with ammonium sulfate at rank 1

Organic moleculesb 13 Compounds, with 2-methyl-2,4-pentanediol at rank 1

Polymers 10 Families, notably poly(ethylene glycol) (first use in 1976), Jeffaminesc,d(1992),

poloxamersc,e(2009), miscelleanous polymersc(2010), polysaccharidesc,f (2011) with

poly(ethylene glycol) 6000 at rank 1

Ionic liquids Imidazolium-based compounds (first use in 2007) after a precursory finding in 1999 on

the properties of ethylammonium nitrate

Buffer and pHb High success rate in the pH 6–8 range and near pI of proteins

Supersaturationb Controls nucleation (number of crystals)

Ligands Modify macromolecules properties (importance of stoichiometry)

Additives Metal ionsb; other ions; miscellaneous small compounds (in mM range)

Detergents > 50 Potentially useful detergents for membrane proteins; can be useful for ‘soluble’ proteins

Physical

Purity Beneficial effects of conformational purity; solid impurities (dust particles)

Temperatureb Tested in the range 4–60 °C; temperature-dependent solubility; temperature fluctuations

Time Minutes to years for nucleation; can modify properties of macromolecules

Pressure (up to 220 MPa) Affects solubility and nucleation (first tested in 1990)

Magnetic field (up to 10 T) Diminishes convection, can orient crystals (first tested in 1997)

Electric field (up to 10 kV�cm�1) Affects nucleation rate (first tested in 1999)

Gravitational fieldg

Earth gravity (1 g) Importance of convection & sedimentation at 1 g

Microgravity (10�6 g range) In space shuttles, stations or satellites; frequent g-variations during flights

Hypergravity (> 1000 g) In ultracentrifuges (from 1000 to 40000 g) (first tested in 1936)

Flow and motion Hampers or enhances crystal growth (combined effects of convection and diffusion)

Minimized In gelled or viscous media (microgravity mimicry), first tested in 1954

Enhanced/amplified By deliberate stirring (first explicitely tested in 2002)

Vibrations and soundsc Mostly uncontrolled; also deliberate sonocrystallization proceduresc

Laser irradiationc Triggers nucleation by cavitation effect (first tested in 2006)

Geometry of set-ups Influences crystallization kinetics (equilibration) (see text)

Volume and geometry of samples Affects physico-chemical properties of sample media (see text)

Contact surfacesc Heterogeneous nucleation and deliberate epitaxy)

Biological

Macromolecule Can be considered as the most important parameter

State Homogeneity; purity; presence of natural contaminants

Origin Extremophiles versus mesophiles and difficulty with Eukarya

In vivo modification Modification of amino acids/nucleotides; enzymatic fragmentation

Genetic variants Crystallizability affected by mutations (e.g. disruption of crystal contacts; conformational

changes in the protein)

a For details, see text and crystallization databases (e.g. http://xpdb.nist.gov:8060/BMCD4/index.faces). Most of the crystallization parame-

ters were known in 1990.b Only a few crystallization parameters were explicitly characterized before 1934.c Note the few additional parameters that were identified after 1990.d Jeffamines are polyetheramines based on either propylene oxide (PO), ethylene oxide (EO) or mixed PO/EO backbones with terminal

amino groups.e Poloxamers are amphiphilic non-ionic multiblock polymers.f Polysaccharides include alginic acids, chitosans, pectins and dextrins.g The gravity force is g (on Earth, the standard acceleration due to g is 9.81 m.s�2).



tRNAAsp precipitate in a drop (provoked by spermine)

by adjunction of water in the reservoir that was fol-

lowed by the appearance of a new crystal form of this

tRNA [48]. Vapour-diffusion was followed in the early

1970s by improved dialysis methods with the invention

of new dialysis arrangements (e.g. Cambridge buttons,

Lagerkvist cells, capillaries), initially promoted by pro-

jects on aminoacyl-tRNA synthetase (aaRS) crystalli-

zation [49,50]. At the same time, the free interface

diffusion method became popular [51] and generated

related methods, such as a liquid-bridge variant [52]

and a hybrid method combining dialysis and diffusion

in capillaries. The latter method is attractive because it

allows a decrease in the number of crystal nuclei and

an increase in crystal size by temperature pulses [53].

Of practical interest was the miniaturization of the

batch method to the microlitre level. In that case,

experiments are conducted in sitting drops under oil to

prevent evaporation and to keep volume constant [54].

A common characteristic of these methods is a signifi-

cant reduction of the volume of individual assays that

decreased by ~ 100-fold (from the millilitre- down to

the 2–50-lL range), thereby allowing a more extensive

screening of the parameter space with limited amounts

of macromolecules.

Two methods that were forgotten for a long time

and that have recently been rediscovered are worth

mentioning at this point. The first is protein crystalli-

zation by centrifugation. This was already used in

1936 to crystallize the coat protein of TMV [55] and

its physical basis was investigated in some depth in the

1970/80s with the growth of catalase crystals of vari-

ous sources in a preparative ultracentrifuge at Institute

of Crystallography in Moscow [56,57]. Even though

the original Russian publications were translated into

English, they were overlooked by most western scien-

tists, despite the fact that the centrifuge-grown crystals

led to the first structure of a catalase solved in collabo-

ration with the Rossmann laboratory [58]. The method

was rejuvenated and miniaturized in 1992 with the

crystallization of the Trichoderma resei aspartic pro-

teinase [59] and was thoroughly reinvestigated in 2008

with the crystallization of a panel of model proteins

and a RNA plant virus at hypergravity levels between

1000 and 22000 g [60]. The underlying idea of the

method is to create by centrifugation a gradient of

protein concentration in the crystallization vessel that

encompasses a supersaturated region favourable for

nucleation. A similar rationale underlies a hybrid dial-

ysis method where an increase of protein concentration

Table 4. Early crystallization methods and their variants with examples of deliberate crystallizations for X-ray crystallography. For

crystallization data, see http://xpdb.nist.gov:8060/BMCD4/index.faces. For references, see text and Data S4.

Method Remarks, Cell type (sample volume) Early applications, Year (macromolecule)

Batch methods

Conventional Vials (mL range) 1971 (sea snake erabutoxin)

Jakoby variant Applicable to protein samples of ≥ 4 mg 1971 (proteolyzed E. coli MetRS)

Microbatch Drops under oil (≤ 2 lL) 1990 (e.g. lysozyme)

Dialysis

Conventional Dialysis bags (> 1 mL) 1959 (e.g. yeast cytochrome b2)

Microdiffusion Zeppezauer cells (≤ 100 lL) 1968 (e.g. lysozyme)

Heavy-walled capillary cells (≤ 100 lL) 1970 (e.g. aldolase)

Meso and micro methods Lagerkvist cells (~ 50 lL) 1972 (S. cerevisiae LysRS)

Cambridge cells (4–350 lL) 1973 (B. stearothermophilus TyrRS)

Microcaps (< 50 lL) 1985 (E. coli enterotoxin)

Double-dialysis Cambridge buttons 1989 (Staphylococcus aureus delta toxin)

Interface diffusion

Conventional free interface

method

Pasteur pipettes and other types of glass tubes

(diameter < 6 mm)

1972 (validated with several proteins,

e.g. cytochromes)

Liquid-bridge variant Droplets of protein sample and mother liquor

connected by a liquid-bridge

1974 (Chlorobium limicola bacteriochlorophyll-

protein)

Hybrid diffusion/dialysis

method

Capillaries submitted to temperature pulses 1975 (Lactobacillus casei thymidylate synthetase)

Vapour-diffusion

Sitting drop Plates/trays with 6–100 drops (2–40 lL) 1968 (S. cerevisiae tRNAPhe)

Hanging drop Tissue culture plates with 24 wells (2–20 lL) 1971 (carp albumins)

Sandwiched drop Drops between two glass plates 1994 (bacterial cytochrome C-552)

Capillary apparatus Sample in a capillary (≤ 1 lL) 1988 (ribosome)



occurs by electrophoresis [61]. The second forgotten

method, first published in 1954 [62], is protein crystal-

lization in a gelled medium where convection is

reduced. This diffusion-dependent method, validated

by the crystallization of human serum albumin (dimer

form) in gelatin, was rediscovered 34 years later [63] in

the frame of microgravity projects. Other methods

only marginally exploited are crystal growth under lev-

itation [64] and at high pressure [65]. Interestingly, in

all of these atypical procedures, the parameter ‘diffu-

sion’ and hence its counterpart ‘convection’ are on the

forefront (see implications below).

Attempts to control and understand the

crystallization process of biomacromolecules

Before the first interdisciplinary conference on protein

crystallization in 1986 (ICCBM1), attempts to under-

stand the physico-chemical basis of protein crystal

growth were extremely scarce [66]. For example,

Schlichtkrull concluded that, after initial nucleation,

subsequent nucleations occur mainly on the surface of

existing beef insulin crystals [67] and Bunn distin-

guished between amorphous and crystalline material

when measuring the solubility of calf rennin [68]. The

situation changed radically when physicists outside of

biology entered the game and tried to adapt the theo-

retical background of small molecule crystal growth to

the protein field [69–71]. This trend was also fostered

by the first protein crystallizations in microgravity [72–74]. As a result, data accumulated rapidly and signifi-

cant information was obtained with model proteins

(lysozyme, canavalin, concanavalin A) on precrystalli-

zation [75–77], nucleation [78–83], growth [78,79,81,84]

and cessation of growth [79,80,83]. For example,

nucleation rate and final lysozyme crystal size were

found to depend upon the rate at which critical super-

saturation is approached [81].

The establishment and exploration of phase dia-

grams represented important trends (Fig. 2A). Initial

investigations were conducted on nucleic acid crystals

grown by the vapour-diffusion micromethod; first of

yeast tRNAPhe [85] and, subsequently, of DNA frag-

ments [86]. The combined effects of Mg2+ and sper-

mine concentrations on crystal quality were explored

and, in the case of tRNA, this allowed the identifica-

tion of a crystal polymorph diffracting at high resolu-

tion [85]. In the protein field, initial investigations were

conducted on lysozyme by material-consuming batch

methods (1–80 mg of protein per measurement), show-

ing the rate-limiting attachment of protein molecules

on growing crystals, the preferential growth of large

crystals from moderately saturated protein solutions

[87] and temperature-dependent solubility accompanied

by negative or positive crystallization heats for tetrago-

nal or orthorhombic polymorphs [66]. Because high

amounts of material were needed refrained to explore

phase diagrams, this encouraged the development of

user-friendly micromethods based on vapour-diffusion

in 10-lL drops and of sensitive microassays for mea-

surement of protein concentration. This allowed sys-

tematic studies with Arthrobacter glucose isomerase,

jack bean concanavalin A and HEW lysozyme. Thus,

glucose isomerase crystallization was found to be pH-

A B

Fig. 2. Phase diagrams. (A) Theoretical 2D phase diagram showing how macromolecule concentration relates to crystallant concentration;

the diagram is multidimensional and can encompass additional dimensions covering physical parameters (see list provided in Table 3). The

diagram shows how the solubility curve separates the undersaturated region with the three zones of the supersaturated region and also

how parameters vary in a crystallization assay (from the undersaturated region toward the nucleation zone following trajectory a, until a

nucleus forms in b that will grow following trajectory c until the crystal/solute equilibrium is reached in d) [14]. (B) Part of the phase diagram

of concanavalin A from jack bean showing the solubility of the lectin as a function of ammonium sulfate concentration and temperature [82].



dependent over pH range 5.5–6.5 [88]. With concanava-

lin A, solubility decreased when salt concentration

increased in accordance with the empirical Green’s law

(see above) and increased with temperature (Fig. 2B).

Moreover, crystal morphology was found to be temper-

ature dependent [82]. Importantly, as found for HEW

lysozyme, the main effects of salts on protein solubility

were a result of anions ranked in the reverse order of

the Hofmeister series (SCN� > NO3� > Cl� > cit-

rate2� > acetate� ~ H2PO4� > SO4

2�) [29]. On the

other hand, the discovery of the peculiar effects of

ammonium sulfate at high concentration was

unexpected and was beneficial for the crystallization of

the yeast AspRS:tRNAAsp complex [42,89,90].

In summary, many different crystal forms were

observed when exploring the parameter space of

A B C D

E F G H

I J K L

M N O P

Fig. 3. A gallery of crystals illustrating shape and habit variability, as well as growth pathologies, as observed under different experimental

conditions. (A–H) Crystals of model proteins: (A–D) Diversity of lysozyme crystals grown with NaCl as the crystallant [from HEW: (A)

microcrystalline precipitate, (B) twinned embedded crystals, (C) classical tetragonal habit obtained at high pressure (50 MPa); from TEW: (D)

hexagonal prisms obtained in agarose gel under 75 MPa pressure (length increased and width diminished)]. (E–G) Example of three habits

of jack bean concanavalin A crystals found in a phase diagram screening solubility as a function of ammonium sulfate concentration (0.4–

2.0 M), pH (5.0–7.0) and temperature (4–40 °C): (E) the typical form grown under almost all conditions, (F) round-shaped crystals grown

especially at 12 °C and (G) small crystals growing out of the fracture of a large crystal by 2D nucleation, as occasionally observed in 10 lL

sitting drops. (H) Tetragonal bipyramidal crystals of T. daniellii thaumatin grown in free interface diffusion reactors after 10 days of

microgravity at 20 °C with 1.6 M Na tartrate as the crystallant [USML-2 (United States Microgravity Laboratory-2) mission in October 1995;

note the increased number of smaller crystals at the crystallant entrance of the crystallization chamber at the right side and the gravity

vector from right to left]. (I–P) Crystals of key partners in translation: (I) An orthorhombic yeast tRNAAsp crystal that was useful for structure

determination. (J–L) Crystals of yeast and T. thermophilus AspRS: (J) tetragonal crystals from the yeast enzyme showing growth defects

together with brush-like spherulitic needle bunches and (K, L) gorgeous crystals of the bacterial enzyme from T. thermophilus grown (K)

under microgravity or (L) on earth from a microcrystalline precipitate by Ostwald ripening. (M) Crystals of yeast initiator tRNAMet with

growth pathology not suitable for X-ray analysis. (N–P) Crystalline diversity in yeast tRNAAsp:AspRS complex crystals grown in the presence

of a high concentration of ammonium sulfate, showing a great sensitivity of enzyme purity and RNA/protein stoichiometry: (N) spherulite-like

bodies observed with heterogeneous AspRS and a slight stoichiometric excess of tRNA (spherulites are circular bodies composed of thin

crystalline and divergent needles/fibres), (O) polymorphism in the same crystallization drop showing cubic and orthorhombic crystal habits

and (P) orthorhombic P212121 polymorphs diffracting up to 2.7 �A. For references, see text and S5.



crystallization. A few examples from the author’s lab-

oratory are shown in Fig. 3. The important

conclusion to emphasize at this point was the absence

of a positive correlation between apparent perfection

of crystal habits and high diffraction quality.

From another viewpoint, the breakthroughs brought

by light microscopy, electron microscopy [and later by

atomic force microscopy (AFM), see below] and

dynamic light scattering (DLS), either to visualize and

quantify protein crystal growth processes or as a tool

for crystallization diagnostics, were important. Thus,

monodispersity of protein solutions under precrystalli-

zation conditions, as monitored by DLS, was shown to

be a good indicator of crystallizability [75–77]. Also of

fundamental importance were investigations on lyso-

zyme crystallization that monitored the size and shape

distribution of small aggregates appearing during pre-

nucleation and kinetic features characterizing the

growth and cessation of growth phases [79]. These were

concluded later for non-uniform growth over time

accompanied by imperfections on fast-growing faces

[80] and growth by lattice defects at low supersatura-

tion and two-dimensional (2D) nucleation at high

supersaturation [83]. On the other hand, the time-

dependent pH changes that can occur in vapour-diffu-

sion set-ups [91] and the dramatic variations in water

equilibration rates when varying temperature and ini-

tial drop volume [92] confirmed the importance of

kinetic effects in protein crystallization.

Towards better and optimized crystallization

strategies

The initial efforts towards rationality in protein crystal

growth and the many observations gathered during

empirical practice of crystallization in the 1970s and

1980s led to new concepts (notably on purity) and tech-

nologies for apprehending protein crystal growth, to

the search for optimization strategies, and to proposals

regarding improved crystallization strategies that were

developed in the 1990s (see below). The fact that many

proteins remained recalcitrant to crystallize also stimu-

lated work on the physical chemistry of protein crystal-

lization and the search of biology-based strategies.

A reasonable assumption made by investigators

working with proteins recalcitrant to crystallize was to

conjecture that evolution has shaped more stable pro-

teins in organisms adapted to live under extreme con-

ditions. The idea was validated with a thermophilic

TyrRS [50] that yielded better crystals than the meso-

philic counterparts. The same is true for thermophilic

ribosomes [93]. Rationalization of the concept of

purity was another accomplishment. It was based on

personal observations (e.g. the presence of microheter-

ogeneities in tRNA and protein samples) [94] and data

from literature (e.g. beneficial effects of purification on

both crystal growth and crystal quality) [95–97]. Alto-

gether, this led to a refined definition of what is really

protein purity, namely chemical and conformational

homogeneity, an absence of protein and small mole-

cule contaminants, and stability over time. Consider-

ations about purity gave also a refined view on the

nature and importance of impurities (isoforms or

denatured/aggregated versions of the protein of inter-

est, foreign protein material, small molecule contami-

nants) in protein samples that could affect

crystallization. Striking examples concern contami-

nants present in poly(ethylene glycol), especially phos-

phate or sulfate anions [accounting for the growth of

Eco elongation factor polymorphs depending on the

brand of poly(ethylene glycol) used] [98] or aldehydes

and peroxides that were shown to affect the crystalliza-

tion of rabbit muscle phosphoglucomutase [99]. In this

context, a crystallization method combining purifica-

tion and protein conditioning in crystallization media

[100] is worth mentioning.

Accordingly, it was conjectured that the intrinsic

flexibility of peptides and many proteins would be det-

rimental to their crystallization. A remedy would be to

stabilize the unstable structures with other macromo-

lecular partners. The idea was validated with the crys-

tallization of antibody:antigen complexes, with initial

proof-of-concept experiments using lysozyme as the

antigen [101]. The relative ease to prepare monoclonal

antibodies permitted a rapid generalization of the

strategy with, for example, the crystallization of neur-

aminidase from influenza virus [102] or of the human

angiotensin II peptide [103] in complex with specific

Fab fragments derived from monoclonal antibodies.

Today, cocrystallization strategies have many applica-

tions in structural biology (see below).

Among thermodynamic parameters, temperature

and time [104] were identified first as being important

for protein crystallization. Both affect protein confor-

mation and, consequently, solubility, as well as crystal

habits (Fig. 3) and growth mechanisms. Similar effects

are brought about by pressure [65] and pH changes

[104]. On the other hand, nonreproducibility remained

a major drawback and pointed to the primordial role

of the geometry and size of set-ups (both crystalliza-

tion chambers/drops and reservoirs) that affect equili-

bration kinetics and modulate the balance between

convective and diffusive mass transport during crystal

growth, as well as the extent of crystal floating or

sedimenting in the mother liquor. Furthermore, exper-

imental evidence indicated heterogeneous and epitaxial



nucleation brought about by contact of proteins with

solid surfaces [105], with such phenomena even occur-

ring on the surface of growing crystals (Fig. 3G). The

fact that diffusion is favoured under microgravity

(and convection disfavoured), as well as the expecta-

tion of better crystals when grown in this environ-

ment, was the main justification of crystallization

programs in weightlessness. Initial experiments show-

ing growth of larger lysozyme crystals [72] were the

start of a race for the access to microgravity [106],

which generated both controversial debate [107] and a

search for an alternate means to favour diffusive mass

transport on earth. This line of thinking was first sug-

gested in 1988 by Robert and Lefaucheux, who grew

lysozyme and porcine trypsin crystals in gelled media

[63], and was largely exploited in the 1990s with stud-

ies of protein crystallization by counter-diffusion or

under magnetic- and electric-fields (Table 3; see also

below). On the other hand, seeding procedures were

recognized as practical means to optimize crystalliza-

tion as soon as initial crystalline material becomes

available. They have been shown to trigger new nucle-

ations or to enlarge the size of crystals [108].

Because of the impressive number of parameters

affecting protein crystal growth and crystal quality,

which largely exceed that involved in small molecule

crystal growth (Table 2), it became rapidly evident

that identifying the appropriate crystallization condi-

tions could not occur by systematic screening of the

parameter space. The need to understand the hierarchy

of parameters and their relationships became essential.

This was not an easy task because this hierarchy is

dependent on the class of macromolecules. Emblematic

examples are the detergents essential for membrane

protein crystallization [43,109] but not required for sol-

uble proteins, although they can have beneficial effects

[110], and the polyamines that are only essential for

tRNA crystallization [111]. To overcome difficulties,

statistical methods were invented. The first comprised

an incomplete factorial method validated with B. ste-

arothermophilus TrpRS that aimed to find correlations

between crystallization parameters and crudely esti-

mated crystal quality [112,113]. It was followed in the

1990s by sparse-matrix methods (see below). In paral-

lel, robotic systems were proposed to facilitate the

practice of crystallization and to achieve better repro-

ducibility [54,114].

Summary before entering the era of structural

genomics

The cooperation between biologists and physicists in

the 1980s with respect to crystal growth, as illustrated

by the first ICCBM Conferences, provided insights

into the mechanistic aspects of protein crystal growth.

On the other hand, crystallization was no longer

restricted to isolated proteins and now also concerned

protein assemblies, nucleic acids and nucleic acid:pro-

tein complexes. Highlights were the miniaturization of

conventional batch and dialysis crystallization and also

the invention of vapour-diffusion methods. Vapour-

diffusion methods were rapidly adopted by structural

biologists because of their versatility, although draw-

backs were soon intuitively recognized. They rely on

the fluctuating geometry of the crystallizing drops and

the dynamic nature of the vapour-diffusion process

leading to a decrease of protein concentration and a

concomittant increase of impurities in the crystallizing

media, accompanied by an enhanced poisoning of the

growing crystals, as first suggested by Wayne Ander-

son [115]. Because physico-chemical conditions in crys-

tallization drops are not well defined, this might

explain the large number of irreproducible results.

Batch methods that are more static and easier to

implement remained popular, especially in their minia-

turized versions under oil. Despite the remaining poor

understanding of many aspects of protein crystalliza-

tion, the sound theoretical basis that emerged in the

period between 1934 and 1990 opened new routes for

more rational and efficient biocrystallogenesis, which

were successfully explored in the era of structural

genomics.

Crystallogenesis in the era oftechnologies and structural genomics(1990–2013)

Crystallogenesis always benefited from the interplay

between science and technology. This trend became

especially prevalent after 1990 when the new biotech-

nologies provided tools for the preparation of any

type of protein or nucleic acid present in nature and,

when new instrumentations and robotic systems

became accessible, for more efficient crystal growth

and faster crystal analysis [116]. The initial fundamen-

tal work with HEW lysozyme and a few other model

proteins was pursued, and extended to membrane

proteins and a large panel of other proteins and mac-

romolecular assemblies of high biological value. In

parallel, ideas originating from fundamental work

were translated into applications useful for the growth

of crystals for structural biology. Altogether, this led

to a paradigm change with deep impact upon the

field. New strategies for qualitative and quantitative

evaluation of the different steps of the crystal growth

process were proposed and specific crystallizability



features were discovered. The need for large crystals

declined with the easy access to second-generation

synchrotron X-ray sources, a trend that even applies

for modern neutron crystallography. Automation pro-

gressively became essential in crystallogenesis and,

recently, the nascent nanotechnologies found many

applications in structural biology. Last but not least,

the biocrystallogenesis field received support from

Space Agencies that fostered microgravity research

and were particularly interested in protein crystalliza-

tion. Altogether, during the period between 1990 and

2013, the field benefited from dramatic advances in

analytical and gene technologies and was nourished

by a constant interplay between fundamental and

practical focused research. For simplicity, these two

aspects are discussed separately.

Fundamental crystallogenesis

The effects exerted by physical and chemical parame-

ters on macromolecular crystal growth and many

related questions have been investigated in depth by a

variety of approaches [117,118]. Exploration of param-

eter-space in phase diagrams was first on the forefront

for the selection of parameters leading to protein crys-

tallization. Imaging growth processes, scrutinizing

crystal anatomies and, important from the viewpoint

of structural biology, comparing X-ray structures

solved from crystals grown under different conditions,

represented other challenges. In the late 1990s, investi-

gations on atypical physical, chemical and method-

related parameters that might affect crystallization

became more prevalent and led to alternative crystalli-

zation methods. This was first the case for micrograv-

ity and related factors and, more recently, for light,

ionic liquids, new additives, the volume of crystallizing

samples and the geometry of set-ups. Other goals were

to control nucleation and to uncouple it from growth.

Solubility and phase diagrams

Because of the multiparametric nature of the crystalli-

zation process, protein phase diagrams are multidi-

mensional and therefore can only be partly explored.

Their landscapes represent the solubility behaviour of

proteins under crystallization conditions and can be

considered as footprints characterizing individual pro-

teins or group of proteins. Thus, the proper handling

of these parameters could be used to initiate and con-

trol crystallization. Based on empirical rules derived

from Arda A. Green’s work [39,40] and precursory

theoretical thoughts on protein solubility, it was

expected that some general rules could govern protein

solubility and thus predict crystallization. However, as

a result of a poor understanding of the crystallization

process, only qualitative rules could be expected at the

time. Thus, the pH-dependent solubility of proteins,

with a minimum at the isoelectric point (pI), where the

average charge is zero, is accounted for by the zwitter-

ionic nature of proteins. Similarly, the salt-dependent

solubility relies on the ionic interactions that salts can

make with proteins. This occurs especially at high

ionic strength as reflected by salting-out (i.e. decrease

in solubility when the salt concentration increases) and

at the less frequent opposite and poorly understood

salting-in (i.e. increase in solubility) phenomena.

Understanding how the many other factors listed in

Table 3 affect protein crystallization remained essen-

tially unknown, notably the gravity-related factors

convection and diffusion, which are well explored for

the crystal growth of conventional molecules but not

for that of proteins [69,119].

To obtain insight into these unexplored issues, sys-

tematic studies were initiated in the early 1990s, first

on the effects of Hofmeister salt concentrations, pH

and temperature on protein solubility and crystal

growth, and later on those of a variety of additional

parameters, either chemical [organic crystallants such

as poly(ethylene glycol)] or physical (pressure, convec-

tion, diffusion, light, etc.). Experiments were con-

ducted not only with the standard models, but also

with proteins of interest for structural biology. Thus,

besides exploring crystallizability of HEW lysozyme

[120–123], partial phase diagrams were established,

amongst others, for a collagenase [124], two membrane

proteins (bovine cytochrome c, Rhodobacter sphaero-

ides photoreaction centre) [125,126], a carboxypepti-

dase [127], S. cerevisiae AspRS [128] and even plant

viruses [129,130]. Thus, with lysozyme and whatever

the pH, increasing pressure resulted in greater numbers

of crystals, as well as a transition from the initial

tetragonal to the orthorhombic polymorph [122]. On

the other hand, huge temperature effects on solubility

were observed with most investigated proteins

[131,132]. In the majority of cases, a normal tempera-

ture-dependence was observed (increase of solubility

with temperature, as for lysozyme, trypsin and insulin)

but retrograde solubilities were suggested as well

(decreased solubility, as for catalase and glucose

isomerase) [131]. The situation was paradoxical with

T. daniellii thaumatin known to crystallize with Na

tartrate because the temperature-dependence of its sol-

ubility depends on the chirality of the tartrate ion (i.e.

normal with L-tartrate and retrograde with D-tartrate)

[133]. This observation is of importance because it rec-

onciles the contradictory results obtained with crystals



grown from solutions of racemic Na D,L-tartrate.

Other dramatic temperature effects were observed with

S. cerevisiae tRNAPhe, notably a transition between

three different growth mechanisms within a narrow

range of only 5 °C as seen on AFM images [134].

Regarding the effects of Hofmeister salts on solubility,

these differ globally for acidic and basic proteins and,

in the case of individual proteins, they depend on the

acidic (pH < pI) and basic (pH > pI) state of the pro-

tein [135], as well as the kosmotropic and chaotropic

nature of the salts (making strong or weak, respec-

tively, water interactions in the solvent shell around

the protein).

In the case of the poly(ethylene glycol), often associ-

ated with salts, the situation becomes more complex

because liquid–liquid phase separations are frequently

observed, with consequences on protein solubility

[136,137]. Thus, with the extremelly soluble Aspergil-

lus flavus urate oxidase, a poly(ethylene glycol)-

induced depletion potential in the protein solution

could be demonstrated by small-angle X-ray scattering

(SAXS) measurements and validated by theory [137].

It was also shown that the liquid–liquid phase separa-

tion precedes and slows down crystallization [138].

Globally, poly(ethylene glycol) modifies phase dia-

grams and favours the attractive intermolecular inter-

actions needed for crystallization. This offers the

possibility to control crystallization by varying the size

and concentration of the poly(ethylene glycol) in crys-

tallization media.

Of practical interest were light scattering studies

[both SAXS and small-angle neutron scattering

(SANS)] (Table 5) that established a correlation

between the second virial coefficient B22 and solubility

[139,140]. This coefficient characterizes the nature and

the strength of the interactions between protein parti-

cles in solution and thus provides essential information

on crystallizability. If B22 is positive, the overall intrec-

tions are repulsive. By contrast, if B22 is negative, the

interactions are globally attractive, which favours crys-

Table 5. Diagnostic tools for protein homogeneity, crystallizability and crystal quality. For references, see text and Data S7.

Tool Type of information (year of early inputs)

AFM Growth mechanisms (1992); growth pathologies (1992)

Calorimetry Thermodynamics of crystal growth; stabilization of proteins by additives (1996)

DLS Screening homogeneity protein homogeneity under precrysrallization conditions (1978); detection of

nucleation (1978)

Electron microscopy toolsa Visualization of lattice defects and 2D nucleation (1990); in situ detection of crystalline phase in biological

samples (2002); sample-quality analysis of membrane proteins (2003)

Fluorescence spectroscopiesb Detection of salts in crystals (1997), of protein aggregation in solution (2009)

Interferometryc Quantitative mapping of solution properties (solute concentration, convection, etc.) around growing crystals

(1993)

Informatic predictionsd Incomplete factorial and sampling methods (1979); database screening (2003)

Sequence-based crystallizability prediction (2006); nucleation prediction (2012)

pI (2004)

Mass spectrometry Content of macromolecules in crystals and detection of bound or contaminating small molecules within

crystals (2000)

Optical light microscopiese Crystal habit (1840); protein crystal detection in crystallization media with precipitates (2010, 2012);

measure of growth velocities on individual elementary steps (2012)

PAGE and IEF Sequence size homogeneity (1982); crystallization screening (2001)

Raman microscopy Quality control of crystals with derivatized proteins (2008)

SANS Time resolved diagnostic of the crystallization process (2008); protein fate in precrystallization (1994) and

supersaturated solutions (1995)

SAXS Crystallization screening (1995), following crystallization process (1998); detection of crystallization artefacts

(2010)

Surface plasmon resonance For identifying compounds that bind to target proteins (2012)

Thermophoresis Search of macromolecule solubility on a thermal gradient device (1998) and crystallization screening (1999)

X-ray topography Visualizing crystal perfection (1996)

a Classical and scanning electron microscopy, transmission electron microscopy, etc.b X-ray, UV and correlation fluorescence spectroscopies, etc.c Mach–Zehnder, Michelson and dual polarization interferometry, etc.d Experimental and virtual bioinformatic predictions, etc.e Classical light microscopy and advanced methods, such as laser confocal differential interference contrast miscroscopy, second-order non-

linear optical imaging of chiral crystals and ultrahigh resolution optical coherence tomography.



tallization, a conclusion that received theoretical sup-

port [141]. From the viewpoint of phase diagrams, the

existence of a metastable liquid–liquid immiscibility

region was predicted in which small liquid droplets

with a high protein concentration form before nucle-

ation proceeds. This region corresponds to the ‘crystal-

lization window’ (–8 9 10�4 < B22 < �2 9

10�4 mL�mol�1�g�2), as proposed by George and Wil-

son [139]. A refinement of this concept proposes a

‘kinetic crystallization window’, independent of the

shape and conformation of the protein [142]. It is

characterized by a kinetic coefficient, fc, defined as the

ratio between the diffusion rate of the protein in solu-

tion and its surface integration rate (based on the

kinetics of protein surface self-assembly at the air/

water interface as evaluated by surface tension mea-

surements). Formation of single crystals is kinetically

favoured in the range 1 < fc < 8 where both diffusion

and integration rates are comparable. This criterion

has been succesfuly verified for several proteins [142].

Nucleation and growth

In the 1990s, the focus was to crystallize recalcitrant

proteins and to enhance quality of crystals not suitable

for structural work. This necessitated fundamental

research and was influenced by space-crystallization

programmes. Indeed, theory claimed that a number of

gravity-dependent phenomena that prevent or perturb

crystal growth on earth are minimized in weightless-

ness (e.g. sedimentation, mass transfer, concentration

gradients and convective currents). The logical conse-

quence is an enhanced quality of space-grown protein

crystals. The expectation received support from the

early space-crystallization experiments, thereby justify-

ing ground-based research aiming to obtain deeper

insight into the mechanisms of protein crystallization

and to optimize the forthcoming microgravity mis-

sions. This also stimulated new research lines aiming

to simulate microgravity conditions on earth and to

develop alternative methods of crystallization (see

below) (Table 6). The main results are summarized

below.

Although it was known that nucleation occurs at

much higher supersaturation than growth and that,

once a nucleus is formed, growth follows spontane-

ously, little was known in the 1990s about its exact

mechanism in the protein world, except that it should

depend exponentially on supersaturation and should

occur preferentially on solid surfaces [70]. The reality

of heterogeneous nucleation of proteins induced by

substances, such as contaminating dust or other solid/

colloidal particles, was rapidly confirmed by experi-

ments [105]. It took more time to unravel the nucle-

ation process itself because two decades of intensive

work were needed [143–146] before a comprehensive

two-step mechanism emerged [147]. One reason for

this is that researchers applied classical nucleation the-

ory to solution crystallization without taking into

account differences between theoretical predictions and

experimental results [148]. Thus, according to the two-

step model, crystalline protein nuclei appear inside

pre-existing metastable clusters, which consist of dense

liquid and are suspended in the solution. Such small-

size nuclei have been visualized by AFM [149]. At high

supersaturation, the nuclei are generated in the spinod-

al regime where the nucleation barrier is negligible.

The solution–crystal spinodal helps to clarify the role

of heterogeneous substrates in nucleation and the

selection of crystalline polymorphs. These ideas pro-

vide powerful tools for the control of the nucleation

process by varying the solution thermodynamic param-

eters [147]. It is essential to note that this two-step

model worked out for proteins appears to apply for all

crystallization processes occurring in nature and indus-

try [148]. This new nucleation scenario could explain

specific effects observed with poly(ethylene glycol)

where liquid–liquid phase separations are often

observed, as well as with various substances or solid

supports known as heterologous crystallization nucle-

ants [105,150]. In the particular case of human hair,

which can act as a heterologous nucleant, it was

shown by confocal fluorescent microscopy that the

protein is concentrated on the nucleating surface, with

a substantial accumulation of protein on the sharp

edges of the hairs cuticles [151].

Controlling nucleation has practical applications. A

simple solution consists of removing uncontrolled solid

nucleants by filtration [152]. This can also be achieved

by counter-diffusion methods [153] or by application

of an electric field [154,155]. Another simple solution

is to eliminate poor quality crystals appearing after

nucleation by manual selection [156]. An alternative

possibility would be to stimulate protein nucleation by

temperature or ultrasounds, as demonstrated for small

molecules [157]. This was achieved in the 1990s for

temperature [158] and, more recently, for ultrasound

waves [159] (Table 6).

Growth of protein crystals highly depends on

supersaturation and on the presence of impurities in

the solute. It is also sensitive to crystal-dependent

parameters, such as structure and defects of crystal

faces, as well as on the bonds established between

growth units. At low supersaturation, crystals pre-

dominantly grow by screw dislocations propagating in

a helical path around lattice defects. At higher super-



Table 6. New advanced and old rejuvenated methods of protein crystallization. For references, see text and Data S7.

Method Comments on instrumentation and outputs

Proof-of-

concept (year)

Containerless Electrostatic levitation method (air/liquid contacts) for vapour-diffusion; nice crystals 1990

Batch method with floating drops (5–100 lL) under two oil layers

(liquid/liquid contacts); fewer crystals

1990

Epitaxy Many recent advances: epitaxial growth can occur in vapour-diffusion

set-ups, on minerals, on lipid or protein layers, on etched surfaces

1988

Flow-cell Crystallization under well-defined conditions (e.g. for either quiescent

or forced convection), growth under constant protein concentration)

1986

Hybrid methods combining

Microgravity and gels X-ray topographs indicate more ordered thaumatin crystals than the earth control 1999

Gel and oil Can be operated automatically in microbatch technology; improves the

gel acupuncture method; reduces growth rate

2002

Magnetic field and

levitation

Observation of new phenomena for crystallization and dissolution processes 2008

Microgravity and

counter-diffusion

High quality crystals of a lectin grown in Gel-Tube R crystallization kit

flown in Russian Service Module and crystals of several proteins grown

in the dedicated Granada box operated in ESA FOTON mission

2008

Gel and laser pulses Enhancement of nucleation at very low supersaturation 2013

Induced nucleation by

Continuous light Crystallization by Xe-lamp irradiation or by photon pressure produced by a

continuous wave laser

2006

Laser light pulses Cavitation effects essential for induction of nucleation; allowed

crystallization of many proteins, including membrane proteins;

nucleation can be induced at very low supersaturation at gel-solution interfaces

2003

Natural or modified

surfaces

Modified glass or mica surfaces, porous surfaces, organic fibres, etc. 2000

Langmuir–Blodgett

technology

Use of template protein film for growth of microcrystals 2002

Temperature First conducted in a thermonucleator (with local supersaturation control);

adapted for vapour-diffusion, batch and multiwell microbatch with T-gradient

1992

Ultrasound Nucleation of lysozyme after short ultrasonic irradiation (100 kHz and

100 W); reduction of metastable zone and crystal growth at lower supersaturation

2006

Microgravity Batch, dialysis, vapour-diffusion, free interface diffusion in advanced

protein crystallization facility (APCF) and protein crystallization diagnostic

facility (PCDF), counter-diffusion in Granada box; convection

minimized but frequent perturbations by g-jitters

1984

Microgravity features (e.g. reduced convection and favored diffusion, crystal orientation), simulated in/by

‘Ceiling’ geometry A ‘seed’ crystal attached to the top of a growth cell continues to grow in a

diffusion-limited regime; sedimenting microcrystals do not perturb the growing crystal

2009

Counter-diffusion e.g. Granada box; first known as gel acupuncture method; at present generalized use of

capillaries, works with membrane proteins

1993

Electric field Adapted to microbatch or vapour-diffusion; control of nucleation rate and

better quality of HEW lysozyme crystals

1999

Gelled media Classical devices; mass transport restricted to diffusion 1954

High pressure Batch reactors; control of solubility and crystallization 1990

Hypergravity Operated in batch vessel in ultracentrifuge; metastable starting conditions become

supersatureted during centrifugation

1936

Magnetic field Latest advances in superconducting magnets that provide quasi-microgravity conditions:

improvement of crystal quality (resolution, B-factor) observed

1997

Microfluidic Free interface diffusion, nanobatch, counter-diffusion, formulation chips; variety of chips available

(e.g. for visual crystal inspection, initial X-ray screening and high-throughput data collection)

2002

Levitation Crystals obtained under ultrasonic levitation grow at higher rates are fewer and have better

shape and larger size

2012

Reverse vapour-diffusion Operated in any classical vapour-diffusion system; requires gentle drop volume increase by

vapour-diffusion to dissolve protein precipitates (rediscovered in 1995)

1977

Stirring/vibration/flow Rotatory shaker or mechanical vibrator; improvement of resolution and mosaicity of crystals

grown in stirring mode

2002



saturation, they grow by 2D island formation from

2D clusters/nuclei that form randomly on flat regions

on crystal faces. These mechanisms were predicted by

theory in the small molecule field [70] and were

explicitly visualized by AFM images for proteins

(Fig. 4A) [160], RNA [134] and viruses [160]. AFM

revealed also mesoscopic defects, such as stacking

faults, point defects, vacancies at surface protein lay-

ers on crystal faces and other statistical misalignments

[161]. They originate from perturbed growth condi-

tions, which are unavoidable because crystal growth is

accompanied by a decrease in supersaturation in the

mother liquor. This effect is particularly prevalent

with tRNAPhe crystals that show a dynamic change in

growth morphologies induced by even minute temper-

ature changes [134]. Incorporation of impurities or

microcrystals can further affect crystal growth

[161,162] and harm the production of high-quality

crystals assumed to grow at the lowest supersatura-

tion and with a constant growth regime. Uncontrolled

growth conditions likely account for nonreproducibili-

ty of diffraction properties.

Convective solution flow, mass transport and con-

centration gradients play essential roles in crystal

Aa b c

d e

B

Fig. 4. Visualizing microscopic crystal morphology in AFM and X-ray topographic images. (A) AFM images of (a, b) yeast tRNAPhe crystals

seen at two temperatures [134] and (c) T. thermophilus AspRS crystals. (a) Dislocation hillocks on tRNAPhe crystals are formed at 15 °C by

multiple right-handed (left of image), single left-handed (centre of image) and double right-handed screw dislocations (right of image). (b)

Growth by 2D nucleation at 13 °C showing growth and coalescence of islands and expansions of stacks. Formation of a hole caused by

incorporation of foreign particles during the growth of additional layers is shown in the bottom centre of the image. (c) AspRS growth

proceeds by screw dislocation mechanism, as seen on the (100) crystal face. (B) X-ray topographs on (d) TEW lysozyme and (e) T. daniellii

thaumatin crystals [212]. (d) Optical view and schematic drawing of TEW lysozyme crystals (left), reflection profiles of crystals grown from

solution (top left) and in gel (top right) (notice the same full width at half maximum of 6.5 arcsec of the two crystals), and topographs taken

at the top of the reflection profiles plotted for solution and gel grown crystals (bottom left and right, respectively). (e) The same images for

thaumatin crystals grown from solution and in gel. For references, see text and Data S6.



growth. According to theory, crystal quality is usually

better under diffusion-limited growth where a depleted

zone of the solution surrounds the growing crystal

[70,157]. However, because of convective fluid

motions, the depletion zone is hardly maintained

around a crystal on earth, which might explain why

protein crystals should be of higher quality when

grown under microgravity conditions. To explore these

issues, interferometric studies were undertaken under

earth-gravity and reduced space-gravity. The first data

on protein crystals were obtained in 1993 [163,164]

and were followed by a series of investigations using

Michelson or Mach–Zehnder interferometry that quan-

titatively characterized concentration gradients, deple-

tion zones and diffusion boundary layers around

growing and dissolving protein crystals [161,165–168].From all these studies, it was possible to propose

kinetic models of growth and to realize that quasi-sta-

ble depletion zones form around growing crystals in

space and, consequently, that best conditions for crys-

tal growth occur under microgravity and that vapour-

diffusion geometry does not provide spatially stable

crystal position or fluid conditions for optimized

growth under a diffusive regime. This last conclusion

is in line with other observations made about difficul-

ties encountered in vapour-diffusion methods, as a

result of drop size and shape, geometry of the crystalli-

zation set-up and associated evaporation kinetics that

all control the output of crystallization trials [92,169–171], and accounts for the nonreproducibility likely

explained by uncontrolled physics inside droplets.

A few comments about Ostwald ripening and mem-

brane protein crystallization are worth noting. Ripen-

ing concerns the fate of precipitates occuring at a high

supersaturation that occasionally transform into large

crystals. In the macromolecular field, the phenomenon

was first explicitly described in 1996 for Thermus ther-

mophilus AspRS [32] (Fig. 3), although it has been

occasionally seen by many protein crystal growers. A

recent study shed some light on the mechanism. Using

a combination of DLS, optical microscopy and micro-

fluidics, it could be shown that a dense amorphous

phase constituted by precrystalline protein clusters dis-

plays classical Ostwald ripening growth kinetics but

deviates from this trend after nucleation of the crystal

phase. It was concluded that this behaviour arises

from a metastable relationship between the clusters

and the ordered solid phase [172].

Regarding the mechanism underlying membrane

protein crystallization, although it likely follows gen-

eral rules demonstrated for soluble proteins, it presents

specific features as a result of the intricate interaction

networks created under crystallization conditions by

the detergents, amphiphiles, crystallants and hydro-

phobic membrane proteins. Thus, using SANS, it was

shown that optimization of micelle size and shape for

crystallization requires specific combinations of deter-

gent, amphiphile and crystallant [173]. It was also

shown that poly(ethylene glycol), often included in

crystallization media for membrane proteins, favours

solution conditions where the stability of the liquid

phase changes from stable to unstable [174]. A great

breakthrough came in 1996 when Ehud Landau and

Jurg Rosenbusch replaced the micellar crystallization

media with lipidic cubic phases [175]. These are gel-like

lipid–water systems comprising lipidic compartments

interpenetrated by aqueous channnels that were dis-

covered in the 1960s by Vittorio Luzzati [176]. Recent

data indicate that nucleation of bacteriorhodopsin

crystals occurs in such media following local rear-

rangement of the highly-curved lipidic cubic phase into

a lamellar structure mimicking the native membrane in

which the crystals will grow in a constrained environ-

ment surrounded by lamellar structures [177]. This

mechanism leads to an absence of dislocations and the

generation of new crystalline layers at numerous loca-

tions, as well as to voids and block boundaries. The

characteristic macroscopic lengthscale of these defects

suggests that the crystals grow by attachment of single

molecules to the nuclei [177]. At present, the in cubo

method is widely used [178] and applications for solu-

ble proteins are expected. Recently, the method was

extended to other mesophases in the lipid (monoolein)/

water diagrams and led to a user-friendly fast screen-

ing technology [179].

Microgravity and related issues

A further step towards understanding protein crystalli-

zation consisted of an evaluation of the parameters

governing mass transport and dynamic flow during the

process. Viscosity and gravity are the major parameter

accounting for convection/diffusion and buoyancy-

induced phenomena. Their effects are well known with

respect to the crystal growth of conventional molecules

but were only thoroughly studied in the case of pro-

teins after the first protein crystallization in micrograv-

ity [72] and the claim that reduced convection under

such conditions should favour crystal quality [106]. It

took approximately two decades to convincingly vali-

date the expectation [180]. Overall, space-grown crys-

tals grow larger, and have more regular external

morphology and better internal order with reduced

mosaic spread [181,182], although contradictory results

have been reported [14]. Resolution is sometimes over-

whelmingly improved, as for space-grown paralbumin



crystals that diffract at 0.9 �A, whereas the earth con-

trols are not suitable for diffraction analysis [183]. In a

few cases, when real ground controls were available,

space-grown crystals gave more accurate structures

(e.g. obtained with better defined initial electron den-

sity maps) [180,184,185].

Access to space is not easy and, already in 1988, a first

solution to simulate microgravity effects was proposed

consisting of crystallization in gelled media [63]. This

possibility was followed by proposals advertising pro-

tein crystallization by counter-diffusion [186], under

magnetic [187] or electric [188] fields, and, most promis-

ingly, under microfluidic conditions [189] (Table 6).

Preferential orientations of crystals were observed under

a magnetic field [190] and numerical predictions

revealed the damping of convection by magnetization

[191]. It was also realized that some atypical methods

could reproduce any potential beneficial features of the

microgravity environment, such as crystallization in

containerless systems [192], under hypergravity or at

high pressure.

In all of these methods, mass transport and fluid

movements are affected, as accounted by the dimen-

sionless Grashof GrN number [193], a classical predic-

tor in fluid mechanics, which, in the present case,

evaluates how buoyancy and viscosity forces affect pro-

teins in their liquid crystallization media according to:

GrN� buoyancy forces/viscosity forces�L3 a Dc g m�2

where L is the characteristic length of the system in

which a protein is immersed (e.g. the diameter of a

sphere in which the protein can move), a Dc is a den-

sity gradient dependent on the concentration of the

protein, g is the gravity force, and m is the viscosity of

the fluid. It can be easily seen that the same GrN value

can characterize both microgravity and earth condi-

tions provided that the low gravity force (g) in space is

balanced by adapted geometrical characteristics of the

crystallization device (L) and viscosity forces (m) on

earth. This typically occurs in gelled media and under

counter-diffusion and microfluidic conditions.

A posteriori, the usefulness of these methods for

structural biology is demonstrated by the increasing

number of Protein Data Bank (PDB) entries of struc-

tures solved with crystals grown by these atypical pro-

cedures. In a few proof-of-concept cases, it was shown

that the quality of the X-ray structures solved from

diffraction data originating from crystals grown under

conditions simulating microgravity conditions are

improved. For thaumatin, the crystals grown in aga-

rose gel diffracted to a previously unachieved resolu-

tion and yielded a structure at 1.2 �A resolution

computed from diffraction data collected at room

temperature [194]. In the case of magnetic field, a

comparison of HEW lysozyme structures of 0-T and

10-T crystals revealed only limited overall structural

changes but demonstrated significant fluctuations at a

few residues, improvement in crystal perfection and

increased diffracted intensities leading to a higher res-

olution [195]. Interestingly, for earth- and space-grown

HEW lysozyme crystals grown in the advanced pro-

tein crystallization facility apparatus, counter-diffusion

crystallization even improved the resolution of the

tetragonal crystals from 1.40 to 0.94 �A [196].

The dimensionless Reynold ReN number [193] quan-

tifies the relative importance of inertial and viscosity

forces in fluid dynamics according to:

ReN � inertial forces/viscosity forces � L v/m

where L is the characteristic length of the system, v is

the mean velocity of the protein and m is the kinematic

viscosity of the crystallization medium (m = l/q; wherel is the dynamic viscosity and q is the density of the

fluid). Evaluation of Reynold numbers was used to

find optimal stirring conditions for HEW lysozyme

crystallization [197]. Note that the stirring crystalliza-

tion method is widely used in the small molecule field

for growing high-quality crystals and, in the present

case, it was shown that intermittent flow and low ReNvalues contribute to the growth of a smaller number

of larger crystals [197]. Finally, crystallization of HEW

lysozyme was also analyzed in quiescent and forced-

convection environments [198].

Other theories and simulations predict that shear

flow could enhance or, conversely, suppress the nucle-

ation of crystals from solution. These ideas were tested

in droplets held on a hydrophobic substrate in an

enclosed environment and in a quasi-uniform constant

electric field that induces a rotational flow with a max-

imum rate at the droplet top [199]. The likely mecha-

nism of the rotational flow involves adsorption of the

protein and amphiphilic buffer molecules on the air–water interface and their redistribution in the electric

field, leading to non-uniform surface tension of the

droplet and surface tension-driven flow.

Thermodynamic considerations on protein crystallization

Although thermodynamic approaches are appropriate

to describe solubility, phase separation and crystal

growth processes, they were only scarcely used in the

field of protein crystallization. A first phenomenologi-

cal approach in 1996 with HEW lysozyme found

agreement between values of crystallization enthalpies

determined by calorimetry and by analysis of van’t

Hoff solubility plots [200]. It was followed by a few



other studies [201]. However, a fruitful paradigm

change occurred when researchers tried to understand

the enthalpic and entropic contributions to the Gibbs

free energy of crystallization (DG°cryst = DH°cryst –TDS°cryst) from the viewpoint of chemistry; in other

words, taking into account the contribution during

crystallization of intermolecular bond formation

between protein and solvent. Thermodynamic data

were gathered for several proteins (apoferritin, haemo-

globin C, insulin, lysozyme) and showed that their

crystallization is dominated by entropic phenomena

[202–204]. Thus, the solvent structure, together with

the trapping and release of water molecules, is essential

in the crystallization of these proteins. This implies

structural rearrangements in protein and solvent, mim-

icking by some aspects that which occurs in macromo-

lecular recognition phenomena. These facts have

important consequences for protein crystallization

because, by engineering DS°cryst, it becomes possible to

find thermodynamically favoured crystallization condi-

tions. The idea was exploited under two versions:

either by protein surface engineering to favour inter-

molecular interactions [205] or by calorometry-based

selection of additives for their propensity to stabilize

protein structures in crystallization solutions [206]. A

few proof-of-concept cases of crystal structures of pro-

tein variants showing modified crystal packing contacts

provided strong support for these approaches [205]

(for applications, see below).

Anatomy and quality of protein crystals

Optical microscopy images show a variety of crystal

habits, with some exhibiting perfect shape and symme-

try. However, at higher resolution, as seen by AFM,

crystal faces are not flat but have rough surfaces with

growth-dependent morphologies comprising frequent

imperfections and level differences reaching up to

1000 �A and even more. This raises the important

question of the impact that growth conditions and

growth-induced defects can have on the internal order

of crystals, as ultimately reflected by diffraction prop-

erties. X-ray topography is an appropriate technique

to answer such concerns. It was used for the first time

with protein crystals in 1996 [207,208]. The method

informs about the spread of mosaic blocks, and detects

imperfections and variations in the internal order

within a crystal [209–211]. Typical X-ray topographs

obtained from TEW lysozyme and T. daniellii thauma-

tin crystals grown under two different growth condi-

tions are shown in Fig. 4B. They clearly show,

especially for TEW lysozyme, more homogeneous

images for the gel-grown crystals, demonstrating that

the gel improves crystal quality [212]. Over the years,

the technique has been refined and applied to an

increasing number of proteins. The most recent studies

have characterized individual domains in HEW lyso-

zyme crystals (with homogeneous diffraction qualities)

[213], as well as the presence of loop and curve shaped

dislocations in such crystals [214]. Information on the

internal structure of protein crystals may be useful to

aid in the improvement of crystal growth techniques

[213] and may guide femtosecond laser processing of

gel-grown crystals for diffraction data collection on

the most perfect crystal domain [215].

Summary and main conclusions

The science of biocrystallogenesis has made consider-

able progress in the period between 1990 and 2013, in

great part through the combined efforts of biochem-

ists, biophysicists, protein crystallographers and scien-

tists from the small molecule crystal growth

community. Thus, as anticipated, it was convincingly

demonstrated that the general rules of crystal growth

apply to the protein field. In the case of nucleation, a

novel two-step mechanism was proposed by Peter Ve-

kilov and coworkers that could be generalized for all

crystallization processes, as reported by experts of the

crystal science of conventional molecules [148]. Alto-

gether, a better understanding of the physical chemis-

try of proteins in the different zones of phase

diagrams (Fig. 2A) emerged and, in the case of mem-

brane proteins, understanding how lipidic cubic phases

sustain their crystallization was an important achieve-

ment. From the standpoint of structural biology, it

was realized that crystal growth under diffusive condi-

tions enhances the quality of protein crystals, which is

reflected by the better quality of the crystallographic

models of macromolecules. To reach these conclusions,

a panel of analytical and diagnostic tools (listed with

their characteristic features in Table 5) were of opera-

tional importance. These tools were adapted to the

specific requirements of protein crystallization, in par-

ticular for measurements on microsamples (down to

the microlitre-scale for DLS) [216] or on small and

fragile crystals. The fact that some of these tools are

used by the practitioners of crystallization in structural

biology laboratories is rewarding, especially with

respect to DLS presently being widely used as a diag-

nostic tool regarding protein quality and crystallizabili-

ty [217,218].

Other offspring of the interplay between crystal sci-

ence and technology were proposals followed by vali-

dations of new crystallization methods and an update

of more conventional methods (Table 6). Most of the



novelties exploit atypical parameters (well known for

the crystallization of conventional molecules but not

yet explicitely assayed with proteins, such as tempera-

ture, pressure, stirring) or are based on emerging new

technologies (e.g. the femtosecond laser and nanotech-

nologies). Here also, it is satisfying that practitioners

became progressively convinced of the usefulness of

several of the new crystallization methods. This is

especially the case for counter-diffusion, and gel and

microfluidic based-methods (partly inspired by the

microgravity programmes that created so many con-

troversies among structural biologists), and is reflected

by the increasing number of structures in the PDB

solved from crystals obtained by these methods.

Practical crystallogenesis

This section discusses the changes in the practice of

protein crystallization in the period between 1990 and

2013 and shows how the knowledge gained from basic

research has benefited structural genomics. Different

topics developed synergically (e.g. purity, screening,

structure engineering, high-throughput and automa-

tion, nanotechnology-based methods, optimization)

but, for simplicity, they are discussed separately.

Taken together, a series of strategies for facilitating

and/or enhancing protein crystallization could be

defined (Table 7) and were succesfully employed.

Predicting likelihood of crystallization

Identifying the crystallization conditions of a protein

target can be challenging and explains why research-

ers have tried to relate sample properties with crystall-

izability. This was achieved by exploring the vast

ensemble of data available on macromolular struc-

tures and crystallization features. Although predicting

exact crystallization condition remains a dream,

important guidelines for practitioners originated from

these studies. Thus, predictors of crystallizability were

proposed, with the most emblematic being the second

virial coefficient B22 characterizing undersaturated

protein solutions [217,218]. Also of potential utility is

the kinetic coefficient fc, a predictor reflecting compe-

tition between protein volume transport and protein

surface integration within single crystals or amor-

phous aggregates [142]. Other predictors of crystalliza-

tion likelihood are based on sequence features and

intrinsic physico-chemical properties of the target

proteins (pI, melting temperature, hydrophobicity,

flexibility, etc.) [219–222] or on an analysis of experi-

mentally characterized phase diagrams [223]. For

example, analysis of crystallization data in the PDB

Table 7. A large and diversified panel of crystallization strategies.

For references, see text and Data S1.

Early quotation and year Strategy

Proof-of-

concept

(year)

1971: Creating more compact/less flexible structures

Limited proteolysis 1971

Removal of floppy protein extensions or

fragmentation in structural modules

1994

Chemically synthesized RNA domains 1995

1973: Protein as a variable

Various methods using homologous

proteins with potential better

crystallizability (e.g. from thermophiles,

etc.); screening alternate intrinsic protein

characteristics

1973

1981: Optimization

By seeding procedures 1981

By automation 1990

By controlled drop size variations 2001

By controlled temperature variations 2005

By solubility screening 2005

By advanced DLS methods (e.g. aggregate

size, drop volume., etc.)

2008

By Thermofluor method (estimation of

protein thermal stability)

2011

1983: Cocrystallization with chaperones for soluble and membrane

protein crystallization

Antibody-assisted (antibody fragments

selected by hybridoma or phage display)

1983

Ankyrin-assisted (ankyrins selected by

ribosome display)

2004

1988: Robotics/automation

Use of laboratory robotics to help

crystallization

1987/8

First dedicated system for microbatch

crystallization

1990

Automation of all procedures, in particular

for high-throughput crystallogenesis

2000

1991: Sparse-matrix sampling

A plethora of commercially available

crystallization screens

1991

1991: Protein engineering

Site-directed mutagenesis for modifying

structural properties (e.g. stability)

1991/2

Surface entropy reduction mutagenesis 2001

Site-directed mutagenesis for modifying

physical properties (e.g. solubility)

2012

1992: Uncoupling nucleation and growth

Attempts to control nucleation by a variety

of novel methods

1992

1994: Cocrystallization with chaperones for RNA crystallization

With designed protein modules 1994

With designed RNA module 1998

With antibodies 2011

1998: Heterologous cocrytallization

Easier crystallization if partners originate

from different organisms

1991

For RNA structure determination 1998



revealed a significant relationship between the calcu-

lated pI of successfully crystallized proteins and the

reported pH at which they were crystallized, thus pro-

viding information for the optimal choice of range

and distribution of the pH sampling in crystallization

trials [220]. Another analysis of the PDB indicated

that protein crystals favour some space groups over

others and suggested that symmetric proteins, such as

homodimers, would crystallize more readily on aver-

age than asymmetric monomeric proteins [224]. This

idea was validated experimentally and led to the crys-

tallization of bacteriophage T4 lysozyme after creating

by mutagenesis an artificial homodimer [225]. A

recent attractive tool for crystallization prediction

combines experimentally characterized physico-chemi-

cal features and sequence-derived data from target

proteins [226]. Note that most criteria of crystalliz-

ability are correlated with the fact that the target

should have an enhanced structural stability, as amply

confirmed by many successful crystallization projects

based on this idea (see below).

Biotechnological tools for macromolecule purification

and crystallization purposes

It is common sense, although not always taken into

consideration, that the macromolecule itself is an

important parameter, if not the most important one,

for crystallization, as explicitly discussed for proteins

[227] and nucleic acids [228]. This emphasizes the

importance of purification and macromolecule modifi-

cation procedures in crystallogenesis. In the protein

field, advances towards efficient protein expression

and purification for crystallography are well covered

in the literature [229–231]. However, although DNA

recombinant methods present many advantages, a few

drawbacks detrimental to crystallization should be

noted, such as uncontrolled overexpression leading to

inclusion bodies (particles containing protein aggre-

gates), precipitated and/or denatured proteins, proteo-

lytic degradations, incomplete post-translational

modifications, and so on. Solving these problems can

be time-consuming and costly. Lowering the overex-

pression level represents a possible remedy that

decreases the amount of inclusion bodies. The alter-

nate technology that eliminates most of these draw-

backs is cell-free in vitro protein synthesis. Presently,

only a few crystallography groups have employed this

technology to prepare soluble proteins [232,233] and,

recently, membrane proteins [234]. In the case of

RNAs as well, specific drawbacks have to be over-

come for the preparation of homogeneous samples for

crystallization. They rely on the structural and confor-

mational diversity of RNA molecules and their suscep-

tibility to enzymatic or chemical hydrolytic cleavages.

Although pure RNA samples can be prepared from

crude biological material, enzymatic and chemical syn-

thesis are presently favoured and in case of large

RNAs, enzymatic synthesis using T7 RNA polymerase

is the only possible technology [235]. Note that, for

some RNAs, annealing methods are required to

assume conformational homogeneity [236].

Many methods were used for engineering protein or

nucleic acid variants with enhanced structural stability

favouring crystallization [14]. Limited proteolysis is

probably the simplest one, as already employed in the

1970s [46] and recently rejuvenated in a version where

trace amounts of protease are added/seeded in situ to

crystallization assays [237,238]. Other methods that

aim to refine physico-chemical properties of proteins

were used to specifically favour packing contacts. They

consist of changing surface residues on the targets,

either by DNA recombinant technology [205,239,240],

or by chemical modification [241], in particular by

reductive methylation of lysine residues [242]. These

new methods are based on important precursory

observations, such as the change of a single amino

acid that created a packing contact enabling the crys-

tallization of a human ferritin [243], the application of

the concept of entropy-driven crystal growth of pro-

teins [205], or the idea that intermolecular contacts can

favour or disfavour crystallization and therefore

should be created or eliminated. Also of great poten-

tial is the DARPin technology based on the natural

ankyrin repeat-protein fold with randomized surface

residues allowing specific binding to virtually any tar-

get protein [244,245], thus allowing chaperone-assisted

crystallization.

Producing stable homogenous samples of membrane

proteins for crystallization is particularly challenging

and, as for soluble proteins, screening large numbers

of target proteins is common practice. A new strategy

has recently been proposed that involves the use of

green fluorescent protein fusion constructs and screen-

ing procedures based on expression level, detergent

solubilization yield and homogeneity, as determined by

high-throughput and automated chromatographies

[246]. Notably, antibody-assisted crystallization, intro-

duced by 1983 for soluble proteins [247], applies also

to membrane proteins [248].

Screening crystallization parameters

The idea of using condition screens for the crystalli-

zation of proteins was proposed in 1991 with the

sparse-matrix method [249]. In its original version,



the method used a set of 50 conditions statistically

chosen in a crystallization database to screen the

crystallization of a target protein. After validation of

the method, a rapid release of new screens was

observed, as illustrated by screens of general use

based, for example, on alternate polymeric crystal-

lants [250] and screens specifically designed, for exam-

ple, for RNAs [235,251], protein assemblies [252] or

membrane proteins [253,254]. Today, a large panoply

of crystallization kits is available, either for initial

screening or for optimization [255]. However, many

screens are redundant and making a good choice can

be delicate, especially for challenging projects when

the amount of macromolecular entities is limited and

the number of required trials before success is large.

A new database for the comparison of crystallization

screens could be useful for a rational choice of the

adequate screen [256].

Because the parameter-space for crystallization is

quasi-unlimited, there was always a quest to find new

compounds that sustain or improve protein crystalli-

zation. This quest was pursued in the period 1990–2013 and led to the identification of several classes of

new crystallants, such as Jeffamines, ionic liquids,

poloxamers, polysaccharides and other polymers com-

mercially available, as well as of new detergents.

Among them (Table 3), ionic liquids are particularly

appealing because of the many potential interactions

that they may establish with proteins. Thus, in a pre-

cursory work on lysozyme crystallization published in

1999, it was suggested that the liquid organic salt

ethylammonium nitrate could be of interest for pro-

tein crystallography [257]. It took several years and

more systematic crystallization studies, however,

before the concept could be firmly established [258–260]. On the other hand, the catalogue of additives

that can be of potential use in crystallization trials

constantly enlarges [255], as well as the possible buf-

fers and salt combinations. This creates a huge com-

binatorial diversity of crystallization conditions that

will even augment if the parameter temperature is

included in the screens. Several condition-screening

strategies aiming to restrict the number of trials either

consist of the use of mixes of properly chosen crystal-

lants and/or additives [255] or optimization of the

choice of additives or the buffer formulation by calo-

rimetric approaches [206,261]. Also of practical inter-

est are the positive effects on crystallization of

heterogeneous nucleants introduced on purpose in

crystallization experiments, particularly fragments of

hairs [151], which have their efficacy enhanced when

included in sparse-matrix or high-throughput screens

[262,263].

It should be noted that most compounds within the

screens (except salts) were found empirically and that

their mechanisms of action are not well understood,

especially for the small additive molecules. This is not

satisfactory and does not facilitate the design of effi-

cient new screens. A few recent dedicated studies have

provided some answers with respect to this issue. A

first case study investigated the thermodynamic effects

of acetone on insulin crystallization and concluded

that acetone displaces water molecules on the surface

of the insulin molecules [204]. Another additive widely

used in protein and nucleic acid crystallization, 2-

methyl-2,4-pentanediol, was found bound to proteins

in many crystal structures. Similarly, it could be con-

cluded that binding is accompanied by the displace-

ment of water molecules and promotes stabilization of

the protein molecules, thereby enhancing crystallizabil-

ity [264]. Interestingly, the calorimetric approaches,

discussed above, arrived at the same conclusion

[206,261]. This is in line with the working hypothesis

tested by Alex McPherson, according to which addi-

tives promote crystallization by enhancing intermolec-

ular contacts between proteins or by removing such

contacts between proteins or solvent [241].

Purity and impurities

Purity was a hot topic all along the history of biocrys-

tallogenesis [94] and its versatile importance is con-

stantly emphasized by new publications [265–268]. Forexample, commercial HEW lysozyme used in nucle-

ation studies was shown to contain significant popula-

tions of large pre-assembled lysozyme clusters that

result in a deterioration of the quality of macroscopic

crystals [265]. At the other extreme, lipidic cubic

phase-based crystallizations appear to be more robust

than crystallizations conducted in more classical deter-

gent environments because up to 50% of impurities

are tolerated in the case of the R. sphaeroides photo-

synthetic reaction centre crystals grown in cubic phases

[267]. Similarly, it was shown that highly contaminated

samples of a recombinant Eco protein yielded repro-

ducibly crystals diffracting at high resolution [266].

Given such data, it is understandable that bulk crystal-

lization from impure protein batches remains an open

issue [268].

From the standpoint of applications, it would be

important to understand how impurities can exert

either detrimental or beneficial effects on crystal

growth. When studying the surface morphology of

Bence-Jones protein crystals, it was shown that impuri-

ties adsorbed on the crystalline surface form an impu-

rity adsorption layer that prevents further growth of



the crystal: by growth–dissolution–growth cycles,

impurities can be removed and growth can resume

[269]. In another study, the role of the rate of super-

saturation was highlighted. Thus, when impurity

adsorption on crystal surface is delayed, crystal growth

is enhanced and a ‘purifying’ effect takes place. By

contrast, when impurity desorption is delayed, crystal

‘poisoning’ occurs [270]. This would imply that vibra-

tions, stirring or forced flow during crystallization

[271] could protect from detrimental impurity effects.

Automation and high-throughput

Robotic crystallization systems are efficient, tireless

and accurate, and can carry out experiments using

drop samples of very small volume (1 lL in most

cases, nanolitres in some). They can perform enor-

mous numbers of trials using remarkably small

amounts of biological sample. Many of the robotic

systems reproduce procedures currently used for man-

ual experiments, such as sitting and hanging drops.

They are affordable and well implemented in academic

laboratories [272]. In recent years, and as boosted by

the large Structural Genomics Consortia and Plat-

forms, entire integrated systems have been developed

to accelerate all steps of the crystallization process.

Besides automation of the crystallization trials and

their monitoring, screening of recombinant protein

expression [273], protein purification for crystallization

[274], protein stability [275], image analysis [276], seed-

ing [277] and other optimization procedures [278],

ligand soaking [279], crystal harvesting [280], and crys-

tal mounting [281] have also been automated. More-

over, integrated systems have been installed near to

synchrotron sources enabling in situ diffraction analy-

ses [282]. In summary, automated crystallization by

sparse-matrix methods and screening techniques to

optimize protein homogeneity and crystal quality

improved dramatically and revolutionized the crystal-

logenesis field in the last decade.

It was noted, already one decade ago, that high-

throughput screening of crystallization conditions

does not necessarily produce reproducible results

when carried out in different laboratories, demon-

strating that some important features before crystalli-

zation trials are not under control [283]. This

explains the recent efforts aiming to automatize and

standardize the preparation and handling of samples.

It would also be timely to share worldwide the huge

amount of data generated by the automated high-

throughput crystallization systems with the objective

of extracting useful predictive information. Being

aware of this need, a group a structural biologists

and bioinformaticians convened to develop a crystalli-

zation ontology [284].

Towards nanocrystallogenesis

Scaling-down of crystallization methods was a contin-

uing goal both for practical and theoretical reasons,

aiming at low sample consumption and especially for

the provision of growth conditions favouring crystal

quality. The challenge was to invent miniaturized crys-

tallization devices based either on conventional meth-

ods (batch, vapour-diffusion, etc.) or on alternative

methods that were shown to favour crystal quality

(counter-diffusion, under stirring, etc.) [197,285,286]. A

breakthrough was the demonstration in 2002 of the

feasibility of growing protein crystals in volumes as

small as 1 nL [287]. The same year saw also the entry

of the microfluidic technology in the protein crystalli-

zation field [189]. At the same time, synchrotron tech-

nologies made significant advances (see below) and

offered the possibility of collecting diffraction data on

small crystals [288].

The first microfluidic chip on the market was

based on the free interface diffusion technique [189].

It consists of a complex integrated fluidic circuit

including two networks of channels: one for liquid

handling and a second serving as actuation valves.

The chip was dedicated to high-throughput screening

and was designed to test 48 crystallization conditions

with < 10 lL of sample solution in total. This chip

was modified for the establishment of precipitation

diagrams useful for crystallization screening [289] and

for fine tuning supersaturation in combining free

interface diffusion with vapour-diffusion [290]. A

great step in miniaturization was the possibility to

generate complex mixtures of reagents in 5-nL reac-

tors [289].

Batch crystallization was implemented in a microflui-

dic system in 2003 [291]. In this case, the chip design

was extremely simple and consisted of inlets for pro-

tein, buffer and crystallant solutions, and a microfluidic

channel in which 10-nL droplets are prepared by mix-

ing these solutions in various ratios. This device allows

formulation of thousands of nanodrops, which are car-

ried by a flow of inert oil. The nanodrops can be stored

on the chip and the crystals appearing therein can be

easily analyzed by X-ray diffraction [292]. Based on the

nanodrop approach, a more complex system was

designed for basic research purposes. It is able to for-

mulate droplets and to flow them to storage chambers

where they can be concentrated or diluted by water

permeation through the chamber walls. This PhaseChip

was designed to establish phase diagrams with total



control over supersaturation, nucleation and growth

kinetics in each individual drop [293]. This technology

evolved for measuring nucleation rates [294] and for

manipulating temperature and concentrations in phase

diagrams [295].

Subsequently, counter-diffusion features were suc-

cessfully reproduced in microchannels with the produc-

tion of crystalline material ranging from single crystals

to larger monocrystals along the supersaturation gradi-

ent. When made of the appropriate polymer material,

these counter-diffusion chips allow direct on-chip char-

acterization of the crystals by X-ray diffraction, with-

out any further (and potentially deleterious) sample

handling [296,297].

Another microfluidic technology designated ‘Micro-

capillary Protein Crystallization System’ enables nano-

litre-volume screening of crystallization conditions and

in situ X-ray diffraction studies [298]. The latest

released method is based on controlled evaporation in

the microfluidic device [299]. The many advantages of

the microfluidic chips explain why the microfluidic

technology has become a popular and affordable tool

for various applications, such as condition screening,

optimization, X-ray analysis and basic crystallogenesis

research.

Laser technologies have also been miniaturized and

new laser-based tools for crystal processing have

recently been validated with HEW lysozyme crystals

grown in semi-solid agarose gel and generalized for

other crystals. Processing is carried out using a focused

femtosecond laser, enabling the preparation of small

well cut crystal fragments that are not damaged by the

laser irradiation and are suitable for X-ray analysis

[215]. Such protein microcrystals can be handled by

micromethods [281] and can be used for X-ray studies

by synchrotron microbeam technology [288].

Optimizing protein crystallization methods and crystals

Fabrication of protein crystals suitable for diffraction

studies almost always requires optimization of the ini-

tial crystallization conditions. Seeding is probably the

oldest optimization procedure, as already practiced in

the 1980s [108], and has subsequently been constantly

improved. Seeding techniques (either homogeneous or

heterogeneous cross-seeding with seeds originating

from a different protein) fall into two categories that

employ either macroseeds [108] or microcrystals as

seeds [300,301]. In both cases, the solution to be

seeded should be only slightly supersaturated so that

controlled growth can occur. Several microseeding

methods have been employed, such as streak-seeding

developed by Enrico Stura in the 1990s [300], and

recently automated [302], as well as a microseed

matrix screening method [303], as also automated

[304]. Most recent developments concern, for example,

the adaptation of seeding methods to nanocrystalliza-

tion [305] and the preparation of single microseeds by

femtosecond laser ablation [306].

Most steps and variables in the crystallization pro-

cess can be optimized [307,308] (Table 7). For exam-

ple, this concerns the choice of the crystallization

method. Thus, changing from standard methods to the

counter-diffusion technique improved the crystal of the

core complex of a hydrophobic plant photosystem

[309]. It also concerns the choice of the best tempera-

ture and pH when screening, as well as the size and

volume of the crystallizing samples. Accordingly, tem-

perature cycling [310] and pH optimization [311] strat-

egies have been proposed that were shown to increase

the possibility of obtaining crystals. Another optimiza-

tion technology keeps the crystallization solution meta-

stable during the growth process by controlled

temperature variation of the crystallization solution

[312]. Similarly, it was observed that ultrasound can

optimize nucleation by decreasing the energy barrier

for crystal formation [313]. Furthermore, Thermofluor-

based high-throughput screening methods can be

employed to optimize protein sample homogeneity,

stability and solubility [275].

Optimization also concerns crystal quality, post-

crystallization treatment for enhancing diffraction

quality [314] and crystal size. For a long time, the pro-

duction of crystals of a sufficient size and quality

proved to be a bottleneck in structural investigations.

Although techniques for screening crystals have

improved dramatically, the methods for obtaining

large crystals have progressed more slowly. Despite

many structures were solved from small crystals with

synchrotron radiation, it is far easier to solve and

refine structures when robust data are recorded from

larger crystals. In an effort to improve the size of crys-

tals, a strategy for a small-scale batch method has

been developed, which, in many cases, yields far larger

crystals than attainable by vapour-diffusion [315].

Large crystals are required for neutron crystallography

and, for that purpose, the crystal growth technique

based on temperature variations is particularly appro-

priate [312]. It has been applied to grow high-quality

large crystals of several proteins of interest, which, in

the case of A. flavus urate oxidase, yielded neutron dif-

fraction data in the range 1.9–2.5 �A [316].

Another paradigm change occurred with the advent

of sophisticated X-ray optics, ultrasensitive detectors

and microbeams at new-generation synchrotron

sources [288]. Similar to microfluidic systems, this will



revolutionize the practice of structural biology, with

the consequence that large crystals are no longer a pre-

requisite in X-ray crystallography. Thus, crystals as

small as 20 lm3, corresponding to not more than

2 9 108 unit cells, can yield usable diffraction data

[288]. The same trend, although less extreme, occurred

in neutron crystallography. In that case, a crystal of

0.15 mm3 of perdeuterated human aldose reductase

yielded a structure at 2.2 �A resolution [317]. Impor-

tantly, from the viewpoint of structural biology, smal-

ler crystals are potentially of enhanced quality (see

‘Fundamental crystallogenesis’).

An overall picture of crystallization strategies and their

outputs for biology

Although much remains unclear, the ever deeper

knowledge on crystallization has generated more

rational strategies to produce protein crystals and to

improve their diffraction quality. These strategies are

diverse (Table 7) and have contributed to solving

many bottlenecks in crystallization projects. They illus-

trate how the field of biocrystallogenesis has evolved

in the last 50 years from mainly empirical methods to

sophisticated trial-and-error strategies, as well as to

idea- and basic science-driven methods that slowly

infiltrate structural biology laboratories. Their number

(and the accompanying crystallization methods)

(Table 6) augmented progressively from 1971 until

2013, with a significant boost in the last decade.

Early strategies were based on understanding and

modifying global structural properties of proteins in

view of efficient crystallization; in other words, they

considered the protein, as such, as a parameter affect-

ing crystallization. Thus, simplified and more compact

architectures obtained by proteolysis or genetic engi-

neering, or stabilized by the addition of different types

of structural chaperones, such as antibodies, ankyrins

or macromolecular natural or designed ligands,

showed enhanced crystallizability. This applies to all

types of proteins, including membrane proteins, as

well as RNAs. In that case, the chaperone can be a

general RNA module [318,319] or a protein [320–322].Interestingly, this allowed the opportunity to crystal-

lize biologically significant RNA:protein complexes

[320,322–324].Sparse-matrix sampling combined with robotics

(introduced in the 1990s) played an essential role in

allowing quicker experiments and providing better

reproducibility. Strategies for controlling the physical

chemistry of crystallization were also of prime impor-

tance. They concern uncoupling nucleation and growth

and procedures for optimizing crystallization. As an

example, the screening space of crystallization in

vapour-diffusion methods can be reduced by control-

ling water equilibration, protein solubility and drop

preparation [325]. On the other hand, macromolecular

engineering employed to modify physical properties of

proteins that affect solubility or favour crystal packing

allowed many difficult crystallization problems to be

solved.

In case of difficulties in crystallizing an essential

protein from a given organism, switching to another

organism or, in a more systematic way, screening

orthologues is one remedy [326]. This strategy has

already been employed for proteins [50] and the ribo-

some [93], considering the relative ease of crystallizing

macromolecules from extremophiles, and has been

generalized in a screening procedure of orthologues

[327]. Strategies to optimize crystallization can take

advantage of the large panoply of available crystalliza-

tion methods (Table 6). To date, this potential has

only been partly explored, if not ignored, by practitio-

ners of crystallization, especially hybrid methods com-

bining, for example, crystallization in gel and laser

pulses to induce nucleation or methods based on stir-

ring or vibrations (although vibrations have likely

induced many uncontrolled crystallizations in the

past). Similarly, new devices allowing the growth of

protein crystals in gradient magnetic fields [328] or

assisting with protein crystallization electrochemically

[329] await more thorough testing by practitioners.

Note also an alternative approach of crystallization,

orthogonal to current approaches, developed by Alex

McPherson and colleagues, with the objective of dou-

bling current success rates [330]. It is based on the

hypothesis that many conventional small molecules,

including new crystallants, might establish stabilizing,

intermolecular and noncovalent cross-links in crystals.

Summarizing, it is rewarding to note that the

crytallization toolbox of diagnostic tools, methods and

strategies at the disposal of structural biologists

(Tables 5–7) led to the structure determination of a

variety of important proteins and macromolecular

assemblies (Table 8). Note that many of these successes

are based on recently developed crystallization strate-

gies, such as the femtosecond laser technique [331], mi-

crofluidics [332], the crystallization of complexes with

specific cross-links [333] or hybrid methods [334].

Protein crystals outside crystallography and structural

biology

Although not the subject of the present review, it is

important to note a few applications where knowledge

of protein crystal growth was crucial. This is, for



example, the case with respect to the design of protein-

based biosensors [335]. It also concerns crystallization

for protein purification [336] or safe protein storage

for pharmacological formulations (e.g. the emblematic

example of crystalline insulin) [337]. In this context, it

is worth noting that nature uses this strategy under

certain circumstances to protect macromolecules

against degradation, as is the case of ribosome crystals

found in hibernating animals [338]. More generally,

protein crystals, spherulite-like aggregates and fibres

have been found in vivo in many organisms [339,340],

including in the human body where they are often

associated with severe pathologies, such as Alzheimer’s

and Parkinson’s diseases [340]. Preventing or inhibiting

their formation could therefore have therapeutic appli-

cations. This idea is being explored with respect to the

rational inhibition of amyloid fibril formation [341].

On the other hand, it is known that antibodies can be

raised against protein or small molecule crystals [342].

This opens the possibility of medical applications, such

as for the diagnostic of crystal-based diseases (gout,

Alzheimer’s disease, etc.).

The status in 2013 and perspectives

In 2013, biocrystallogenesis is a mature science based

on strong interdisciplinarity between biology, physics,

chemistry and associated technologies. Today, the

physics and chemistry of protein crystallization are

globally known, although some aspects remain elusive,

Table 8. Examples of emblematic crystallizations based on fundamental or practical advances in protein crystal growth, that led to structure

determination. For references, see text and Data S10.

Year Biomacromolecular particle Crystallization strategy

1968 S. cerevisiae and E. coli pure native tRNA species Conventional method and/or first use of vapour-diffusion (organic solvents as

crystallants)

1980 S. cerevisiae AspRS:tRNAAsp complex Ammonium sulfate as crystallant (most salts disrupt protein:RNA complexes)

1980 B. stearothermophilus ribosome (large subunit) Homologous crystallization (crystallizability of ribosome from thermophiles

better than from mesophiles)

1982 R. viridis photosynthetic reaction centre (first

crystallzation of a membrane embedded assembly)

Ammonium sulfate (crystallant), N,N-dimethyl dodecylamine N-oxide

(detergent) and heptane-1,2,3-triol (additive)

1983 Lysozyme in complex with a monoclonal anti-

lysozyme antibody

Cocrystallization with antibodies

1991 Human ferritin Engineering crystal contacts by analogy with homologous rat ferritin

1994 S. cerevisiae RNA polymerase II Epitaxial growth on 2D crystals on positively-charged lipid layers

1994 Beef mitochondrial cytochrome b-c1 complex Crystals grown in agarose gel

1998 Group II intron domain 5–6 and hepatitis delta virus

ribozyme RNA constructs

Cocrystallization with designed RNA motif

1999 Complex between Eco tRNACys and

Thermus aquaticus elongation factor

Heterologous cocrystallization with partners from two organisms (access to

targeted structure)

2001 Human RhoGDI (cytosolic regulator of GTPases) Protein surface entropy reduction

2002 T. daniellii thaumatin Hybrid method combining microgravity and gel (data collected up to 1.2 �A

resolution at room temperature)

2004 Human aldose reductase Fine biochemistry; crystallization with cofactor and inhibitor (crystals

diffracting at ultrahigh 0.66 �A resolution)

2004 Maltose binding protein and two eukaryotic kinases Cocrystallization with an ankyrin repeat protein

2006 Complex of tRNAGlu and MnmA (an enzyme that

synthesizes 2-thioU at the wobble position of

certain tRNAs)

Femtosecond laser technique

2006 R. viridis photosynthetic reaction centre (proof-of-

concept experiment)

Droplet-based microfluidic batch (at present ≥ 14 solved structures in PDB)

2006 Human glutamate carboxypeptidase II (a large

glycosylated)

Fine biochemistry; heterologous overexpression

2011 Complex of human gankyrin and C-terminal domain

of S6 proteasomal protein

Crystallization of a specific photo cross-linked complex (via incorporation of a

photophore by genetic code expansion)

2012 Thermococcus thioreducens pyrophosphatase Counter-diffusion for neutron crystallography (Hughes RC, Coates C,

Garcia-Ruiz J-M, Blakely M & Ng JD)

2013 Human epidermal growth factor receptor (an apo

cancer-associated mutant)

Hybrid method: microgravity and counter-diffusion

(in JAXA Crystallization Box)

2013 Decameric bacterial SelA:tRNASec ring structure with

heterologous tRNA

Choice of the best bacterial orthologue (Aquifex aealicus) and heterologous

cocrystallization



such as an understanding of the growth of undesirable

protein spherulites in crystallization trials, as appar-

ently favoured by heterogeneous nucleation [343]. It is

expected that the current studies on model proteins

will contibute to finding methods preventing their for-

mation [340,343]. Predicting the likelihood of crystalli-

zation as well has made progress, although

uncertainties still remain in crystallization experiments.

Thus, despite a solid fundamental background, many

protein crystals continue to be obtained by trial-and-

error strategies. However, rational-based crystallization

methods, such as counter-diffusion, as well as crystalli-

zation in gels or nanocrystallogenesis, are slowly being

adopted by crystal growers, and a few others await

more systematic testing, such as stirring methods.

On the other hand, in 2013, the PDB contains

~ 82 000 macromolecular structures solved by X-ray

crystallography (but only 64 structures determined

and/or refined using neutron diffraction data), repre-

senting a large panel of proteins originating from

throughout the tree of life. This could mean that the

bottleneck of crystallization is solved such that, in the

future, protein crystallization will be straightforward.

However, this is not true for three main reasons. First,

the majority of these structures correspond to soluble

proteins and there is a dramatic lack of membrane

protein structures, which are predicted to represent

approximately half of the proteome. Second, the pres-

ently solved RNA structures represent only 3% of the

total and the crystallography of lipids is quasi-inexis-

tent. An increasing awareness of the importance of

RNA and lipids in biology requires a much better

knowledge of their structures. Third, biologists are

becoming more and more ambitious and want to know

ever more intricate and larger macromolecular struc-

tures and assemblies; they especially want to compre-

hend the plasticity and dynamics of proteins and are

even more ambitious regarding macromolecular

machines. In addition, there will always be a need for

structures solved at high and ultrahigh resolution.

Given this situation, one can anticipate further devel-

opments in the crystallogenesis of membrane proteins

[109] and lipids [344], RNAs either free or in complex

with proteins [345] and glycoproteins [346]. Improving

crystallization methods and their application to ambi-

tious biological problems will continue to be at the

forefront of research (e.g. the gel method) [347,348].

This also concerns crystallization on solid nanotem-

plates [349] and other advanced nanocrystallogenesis

methods [350]. Studying crystal polymorphs should

also be pursued and could enable better access to the

structural plasticity of macromolecules, and also erase

possible artefacts resulting from packing effects [351].

From a more global perspective, concepts of mac-

romolecular crowding and macromolecular confine-

ment both in vitro and in vivo [352], should enter

the field of biocrystallogenesis. Thus, one could

question the actual physico-chemical properties of

concentrated protein solutions in nanodrops and

crystallizability (enhanced or inhibited) in crowded

media. Being able to answer such questions could

foster applications for more controlled protein crys-

tallization and, importantly, shed light on in vivo

protein crystallizations and their relation with

pathologies. Moreover, in vivo-grown crystals could

be usable for the emerging technology of free-elec-

tron laser-based serial femtosecond crystallography

[339]. In conclusion, an exciting future is expected

and it is anticipated that the interplay between sci-

ence and technology will continue in the science of

biocrystallogenesis [7].

Acknowledgements

This text is based on lectures given at the FEBS Prac-

tical Courses on ‘Advanced methods in macromolecu-

lar crystallization’, held in Nove Hrady (Czech

Republic) in 2004–2012. It is written to acknowledge

FEBS with respect to its support for the field of bio-

crystallogenesis, which started in 1987 with a FEBS

Lecture Course: namely ICCBM2, in Bischenberg

(France). Warm thanks are extended to Ivana Kuta

Smatanova, Pavlina Rezacova and Rolf Hilgenfeld,

the organizers of the Nove Hrady Courses, to all my

students and coworkers from Strasbourg, past and

present, and to all my colleagues from the biocrystallo-

genesis and structural biology communities for the

exchange of ideas and knowledge over the last

40 years. During all of this period, the support of

CNRS and the University of Strasbourg was essential.

References

1 H€unefeld FL (1840) Der Chemismus in der

thierischen Organisation. Physiologisch-chemische

Untersuchungen der materiellen Ver€anderungen, oder

des Bildungslebens im thierischen Organismus;

insbesondere des Blutbildungsprocesses, der Natur

der Blut K€orperchen und ihrer Kernchen. Ein

Beitrag zur Physiologie und Heilmittellehre.

Brockhaus, Leipzig.

2 Bernal JD & Crowfoot D (1934) X-ray photographs of

crystalline pepsin. Nature 133, 794–795.

3 Gieg�e R & Ducruix A (1992) An introduction to the

crystallogenesis of biological macromolecules. In

Crystallization of Nucleic Acids and Proteins: A



Practical Approach (Ducruix A & Gieg�e R, eds), pp.

1–18. IRL Press, Oxford.

4 Preyer WT (1871) Die Blutkrystalle. Mauke’s Verlag,

Jena.

5 Dounce AL & Allen PT (1988) Fifty years later:

recollections of the early days of protein

crystallization. Trends Biochem Sci 13, 317–320.

6 McPherson A (1991) A brief history of protein crystal

growth. J Cryst Growth 110, 1–10.

7 Gieg�e R (2013) Crystallogenesis at the heart of the

interplay between science and technology in the quest

to comprehend tRNA biology. Cryst Growth Des 13,

405–414.

8 McPherson A (1982) Preparation and Analysis of

Protein Crystals. Wiley and Sons, New York.

9 Gilliland GL & Davies DR (1984) Protein

crystallization: the growth of large-scale single crystals.

Methods Enzymol 104, 370–381.

10 McPherson A (1990) Current approaches to

macromolecular crystallization. Eur J Biochem 189,

1–23.

11 Gieg�e R, Drenth J, Ducruix A, McPherson A &

Saenger W (1995) Crystallogenesis of biological

macromolecules. Biological, microgravity, and other

physico-chemical aspects. Prog Crystal Growth Charact

30, 237–281.

12 Chernov AA (2003) Protein crystals and their growth.

J Struct Biol 142, 3–21.

13 Gieg�e R & Sauter C (2010) Biocrystallography: past,

present, future. HFSP J 4, 109–121.

14 Sauter C, Lorber B, McPherson A & Gieg�e R (2012)

Crystallization. General methods. In International

Tables for Crystallography (Arnold E, Himmel D &

Rossmann M, eds), pp. 99–121. John Wiley and Sons,

Chichester.

15 Russo Krauss I, Merlino A, Vergara A & Sica F

(2013) An overview of biological macromolecules

crystal growth. Int J Mol Sci 14, 11643–11691.

16 Wlodawer A, Minor W, Dauter Z & Jaskolski M

(2013) Protein crystallography for aspiring

crystallographers, or how to avoid pitfalls and traps in

macromolecular structure determination. FEBS J 280,

5705–5736.

17 Hoppe-Seyler F (1864) Ueber die optische und

chemische Eigenschaften des Blutfarbstoffs. Virchows

Archiv Path Anat und Physiol 29, 233–257.

18 Hartig T (1855) Ueber das Klebermehl. Botanische

Zeitung 13, 881.

19 Ritthausen H (1872) Die Eiweissk€orper der

Getreidearten, H€ulsenfr€uchte und €Olsamen.

Beitr€age zur Physiologie der Samen der

Culturgew€achse, der Nahrungs- und Futtermilch.

Cohen & Sohn, Bonn.

20 Osborne TB (1892) Crystallized vegetable proteids.

Amer Chem J 14, 662–689.

21 Guo F, Jin T, Howard A & Zhang YZ (2007)

Purification, crystallization and initial crystallographic

characterization of brazil-nut allergen Ber e 2. Acta

Cryst F 63, 976–979.

22 Osborne TB (2007) The vegetable proteins. In

Monographs on Biochemistry. 2nd edn. (Thomas B

Osborne ed.). pp. xiii+154. London: Longmans Green

and Co. 1924. J Soc Chem Industry 43, 413–452.

23 Funke O (1851) €Uber das Milzvenenblut. Z Rat

Medicin 1, 172–218.

24 Hofmeister F (1890) €Uber die Darstellung von

krystallisirten Eieralbumin und die

Krystalallisierbarkeit von colloider Stoffe. Hoppe-

Seylers Z Physiol Chem 14, 165.

25 Reichert ET & Brown AP (1909) The Differentiation

and Specificity of Corresponding Proteins and Other

Vital Substances in Relation to Biological

Classification and Evolution: The Crystallization of

Hemoglobins. Carnegie Institution, Washington DC.

26 Abel JJ (1926) Crystalline Insulin. Proc Natl Acad Sci

USA 12, 132–136.

27 Hofmeister F (1888) 11. Zur Lehre von der Wirkung

der Salze. Arch exp Path Pharm 24, 247–260.

28 Zhang Y & Cremer PS (2006) Interactions between

macromolecules and ions: the Hofmeister series. Curr

Opin Chem Biol 10, 658–663.

29 Ri�es-Kautt M & Ducruix A (1989) Relative

effectiveness of various ions on the solubility and

crystal growth of lysozyme. J Biol Chem 264,

745–748.

30 Collins KD (2004) Ions from the Hofmeister series and

osmolytes: effects on proteins in solution and in the

crystallization process. Methods 34, 300–311.

31 Ostwald W (1897) Studien €uber die Bildung und

Umwandlung fester K€orper. Z physic Chemie 22,

289–330.

32 Ng J, Lorber B, Witz J, Th�eobald-Dietrich A, Kern D

& Gieg�e R (1996) The crystallization of

macromolecules from precipitates: evidence for

Ostwald ripening. J Cryst Growth 168, 50–62.

33 Sumner JB (1926) The isolation and crystallization of

the enzyme urease. J Biol Chem 69, 435–441.

34 Northrop JH (1929) Crystalline pepsin. Science 69,

580.

35 Northrop JH (1930) Crystalline pepsin: II. General

properties and experimental methods. J Gen Physiol

13, 767–780.

36 Sumner JB & Dounce AL (1937) Crystalline catalase.

Science 85, 366–367.

37 Stanley W (1935) Isolation of a crystalline protein

possessing the properties of tobacco-mosaic virus.

Science 81, 644–645.

38 Kay LE (1986) W. M. Stanley’s crystallization of

the tobacco mosaic virus, 1930–1940. ISIS 77,

450–472.



39 Green AA (1931) Studies in the physical chemistry of

the proteins. VIII. The solubility of hemoglobin in

concentrated salt. A study of the salting out of

proteins. J Biol Chem 93, 495–516.

40 Green AA (1932) Studies in the physical chemistry of

the proteins: X. The solubility of hemoglobin in

solutions of chlorides and sulfates of varying

concentration. J Biol Chem 95, 47–66.

41 Kim S-H & Rich A (1968) Single crystals of transfer

RNA: an X-ray diffraction study. Science 162,

1381–1384.

42 Gieg�e R, Lorber B, Ebel J-P, Moras D & Thierry J-C

(1980) Crystallization of the complex formed between

yeast aspartyl tRNA and its specific aminoacyl tRNA

synthetase. CR S�eances Acad Sci S�er D Sci Nat 291,

393–396.

43 Michel H (1983) Crystallization of membrane proteins.

Trends Biochem Sci 8, 56–59.

44 Zeppezauer M (1971) Formation of large crystals.

Methods Enzymol 22, 235–266.

45 Jakoby WB (1968) A technique for the crystallization

of proteins. Anal Biochem 26, 295–298.

46 Waller J-P, Risler J, Monteilhet C & Zelwer C (1971)

Crystallisation of trypsin-modified methionyl-tRNA

synthetase from Escherichia coli. FEBS Lett 16,

186–188.

47 Hampel A, Labanauskas M, Connors PG, Kirkegard

L, RajBhandary UL, Sigler PB & Bock RM (1968)

Single crystals of transfer RNA from

formylmethionine and phenylalanine transfer RNA’s.

Science 162, 1384–1387.

48 Gieg�e R, Moras D & Thierry J-C (1977) Yeast transfer

RNAAsp: a new high resolution X-ray diffracting

crystal form of a transfer RNA. J Mol Biol 115, 91–96.

49 Lagerkvist U, Rymo L, Lindquist O & Andersson E

(1972) Appendix: Some properties of crystals of lysine

transfer ribonucleic acid ligase from yeast. J Biol Chem

247, 3897–3899.

50 Reid BR, Koch GLE, Boulanger Y, Hartley BS &

Blow D (1973) Crystallization and preliminary X-ray

diffraction studies on tyrosyl transfer RNA synthetase

from Bacillus stearothermophilus. J Mol Biol 80,

199–201.

51 Salemme FR (1972) A free interface diffusion

technique for crystallization of proteins for X-ray

crystallography. Arch Biochem Biophys 151, 533–540.

52 Fenna RE, Matthews BW, Olson JM & Shaw EK

(1974) Structure of a bacteriochlorophyll-protein from

the green photosynthetic bacterium Chlorobium

limicola: Crystallographic evidence for a trimer.

J Mol Biol 84, 231–240.

53 Koeppe RE II, Stroud RM, Pena VA & Santi DV

(1975) A pulsed diffusion technique for the growth of

protein crystals for X-ray diffraction. J Mol Biol 98,

155–160.

54 Chayen NE, Shaw Stewart PD, Maeder DL & Blow

DM (1990) An automated system for microbatch

protein crystallisation and screening. J Appl Cryst 23,

297–302.

55 Wyckoff RW & Corey RB (1936) The ultracentrifugal

crystallization of tobacco mosaic virus protein. Science

84, 513.

56 Karpukhina SY, Barynin VV & Lobanova GM (1975)

Crystallization of catalase in the ultracentrifuge. Sov

Phys Crystallogr 20, 417–418.

57 Barynin VV & Melik-Adamyan VR (1982) The

mechanism of crystallization of proteins in an

ultracentrifuge. Sov Phys Crystallogr 27, 588–591.

58 Vainshtein BK, Melik-Adamyan WR, Barynin VV,

Vagin AA, Grebenko AI, Borisov VV, Bartels KS,

Fita I & Rossmann MG (1986) Three-dimensional

structure of catalase from Penicillium vitale at 2.0 �A

resolution. J Mol Biol 188, 49–61.

59 Pitts JE (1992) Crystallization by centrifugation.

Nature 355, 117.

60 Lorber B (2008) Virus and protein crystallization

under hypergravity. Cryst Growth Des 8, 2964–

2969.

61 Chin C-C, Dence JB & Warren JC (1976)

Crystallization of human placental estradiol 17b-

dehydrogenase. A new method for crystallizing labile

enzymes. J Biol Chem 251, 3700–3706.

62 Low BW & Richards FM (1954) Measurements of the

density, composition and related unit cell dimensions

of some prootein crystals. J Am Chem Soc 76,

2511–2518.

63 Robert M-C & Lefaucheux F (1988) Crystal growth in

gels: principle and applications. J Cryst Growth 90,

358–367.

64 Rhim W-K & Chung SK (1990) Isolation of

crystallizing droplets by electrostatic levitation.

Methods: A Companion to Methods Enzym 1, 118–127.

65 Visuri K, Kaipainen E, Kivim€aki J, Niemi H, Leissla

M & Palosaari S (1990) A new method for protein

crystallization using high pressure. Biotechnology 8,

547–549.

66 Howard SB, Twigg PJ, Baird JK & Meehan EJ (1988)

The solubility of hen egg-white lysozyme. J Cryst

Growth 90, 94–104.

67 Schlichtkrull J (1957) Insulin crystals. V. The

nucleation and growth of insulin crystals. Acat Chem

Scand 11, 439–460.

68 Bunn CW, Moews PC & Baumber ME (1971) The

crystallography of calf rennin (chymosin). Proc R Soc

Lond B Biol Sci 178, 245–258.

69 Rosenberger F (1986) Inorganic and protein crystal

growth – similarities and differences. J Cryst Growth

76, 618–636.

70 Boistelle R & Astier J-P (1988) Crystallization

mechanisms in solution. J Cryst Growth 90, 14–30.



71 Feigelson RS (1988) The relevance of small molecule

crystal growth theories and techniques to the growth

of biological macromolecules. J Cryst Growth 90, 1–13.

72 Littke W & John C (1984) Protein single crystal

growth under microgravity. Science 225, 203–204.

73 DeLucas LJ, Suddath FL, Snyder RS, Naumann R,

Broom MB, Pusey M, Yost V, Herren B, Carter D,

Nelson B et al. (1986) Preliminary investigations of

protein crystal growth using the space shuttle. J Cryst

Growth 76, 681–693.

74 Erdmann VA, Lippmann C, Betzel C, Dauter Z,

Wilson K, Hilgenfeld R, Hoven J, Liesum A, Saenger

W, M€uller-Fahrnow A et al. (1989) Crystallization of

proteins under microgravity. FEBS Lett 259,

194–198.

75 Baldwin ET, Crumly KV & Carter CW (1986)

Practical, rapid screening of protein crystallization

conditions by dynamic light scattering. Biophys J 49,

47–49.

76 Mikol V, Hirsch E & Gieg�e R (1990) Diagnostic of

precipitant for biomacromolecule crystallization by

quasi-elastic light-scattering. J Mol Biol 213, 187–195.

77 Kadima W, McPherson A, Dunn MF & Jurnak FA

(1990) Characterization of precrystallization

aggregation of canavalin by dynamic light scattering.

Biophys J 57, 125–132.

78 Heidner E (1978) Protein crystallizations. The

functional dependence of the nucleation rate on the

protein concentration and solubility. J Cryst Growth

44, 139–144.

79 Kam Z, Shore HB & Feher G (1978) On the

crystallization of proteins. J Mol Biol 123, 539–555.

80 Durbin SD & Feher G (1986) Crystal growth studies

of lysozyme as a model of protein crystallization.

J Cryst Growth 76, 583–592.

81 Gernert KM, Smith R & Carter DC (1988) A simple

apparatus for controlling nucleation and size in

protein crystal growth. Anal Biochem 168, 141–147.

82 Mikol V & Gieg�e R (1989) Phase diagram of a

crystalline protein: determination of the solubility of

concanavalin A by a microquantitation assay. J Cryst

Growth 97, 324–332.

83 Durbin SD & Feher G (1990) Studies of crystal

growth mechanisms by electron microscopy. J Mol

Biol 212, 763–774.

84 Pusey ML, Snyder RS & Naumann R (1986) Protein

crystal growth. Growth kinetics for tetragonal

lysozyme crystals. J Biol Chem 261, 6524–6529.

85 Ladner JA, Finch JT, Klug A & Clark BFC (1972)

High-resolution X-ray diffraction studies on a pure

species of transfer RNA. J Mol Biol 72, 99–101.

86 Malinina L, Makhaldiani VV, Tereshko V, Zarytova

VF & Ivanova Z (1987) Phase diagrams for DNA

crystallization systems. J Biomol Struct Dyn 5,

405–433.

87 Ataka M & Asai M (1988) Systematic studies on the

crystallization of lysozyme. Determination and use of

phase diagrams. J Cryst Growth 90, 86–93.

88 Chayen NE, Akins J, Campbell-Smith S & Blow DM

(1988) Solubility of glucose isomerase in ammonium

sulphate solutions. J Cryst Growth 90, 112–116.

89 Gieg�e R, Lorber B, Ebel J-P, Moras D, Thierry J-C,

Jacrot B & Zacca€ı G (1982) Formation of a

catalytically active complex between tRNAAsp and

aspartyl-tRNA synthetase from yeast in high

concentrations of ammonium sulphate. Biochimie 64,

357–362.

90 Lorber B, Gieg�e R, Ebel J-P, Berthet C, Thierry J-C

& Moras D (1983) Crystallization of a tRNA-

aminoacyl-tRNA synthetase complex.

Characterization and first crystallographic data. J Biol

Chem 258, 8429–8435.

91 Mikol V, Rodeau J-L & Gieg�e R (1989) Changes of

pH during biomacromolecule crystallization by vapor

diffusion using ammonium sulfate as the precipitant.

J Appl Cryst 22, 155–161.

92 Mikol V, Rodeau J-L & Gieg�e R (1990) Experimental

determination of water equilibration rates in the

hanging drop method of protein crystallization. Anal

Biochem 186, 332–339.

93 Yonath A, M€ussig J, Tesche B, Lorenz S, Erdmann

VA & Wittmann H (1980) Crystallization of the large

ribosomal subunits from Bacillus stearothermophilus.

Biochem Int 1, 428–435.

94 Gieg�e R, Dock A-C, Kern D, Lorber B, Thierry J-C &

Moras D (1986) The role of purification in the

crystallization of proteins and nucleic acids. J Cryst

Growth 76, 554–561.

95 Bott RR, Navia MA & Smith JL (1982) Improving

the quality of protein crystals through purification

by isoelectric focusing. J Biol Chem 257,

9883–9886.

96 Spangler BD & Westbrook EM (1989) Crystallization

of isoelectrically homogeneous cholera toxin.

Biochemistry 28, 1333–1340.

97 Van der Laan JM, Swarte MBA, Groendijk H, Hol

WGJ & Drenth J (1989) The influence of purification

and protein heterogeneity on the crystallization of

p-hydroxybenzoate hydroxylase. Eur J Biochem 179,

715–724.

98 Jurnak F (1985) Induction of elongation factor Tu-

GDP crystal polymorphism by polyethylene glycol

contaminants. J Mol Biol 185, 215–217.

99 Ray WJJ & Bracker CE (1986) Polyethylene glycol:

catalytic effect on the crystallization of

phosphoglucomutase at high salt concentration.


100 Gulewicz K, Adamiak D & Sprinzl M (1985) A new

approach to the crystallization of proteins. FEBS Lett

189, 179–182.



101 Mariuzza RA, Poljak RJ, Mihaesco C & Mihaesco E

(1983) Crystals of the human heavy chain disease

protein Riv and human Fc fragment are isomorphous:

further evidence for conformational flexibility in the

hinge region of immunoglobulins. J Mol Biol 165,

559–561.

102 Laver WG, Webster RG & Colman PM (1987)

Crystals of antibodies complexed with influenza virus

neuraminidase show isosteric binding of antibody to

wild-type and variant antigens. Virology 156, 181–184.

103 Garcia KC, Ronco P, Verroust PJ & Amzel LM

(1989) Crystallization and preliminary X-ray

diffraction data of an anti-angiotensin II Fab and

of the peptide-Fab complex. J Biol Chem 264,

20463–20466.

104 McPherson A (1985) Crystallization of proteins by

variation of pH or temperature. Methods Enzymol 114,

125–127.

105 McPherson A & Shlichta P (1988) Heterogeneous and

epitaxial nucleation of protein crystals on mineral

surfaces. Science 239, 385–387.

106 DeLucas LJ & Bugg CE (1987) New directions in

protein crystal growth. Trends Biotechnol 5, 188–193.

107 Leberman R (1985) Crystals in space. Science 230,

373–374.

108 Thaller C, Weaver LH, Eichele G, Wilson E, Karlsson

R & Jansonius JN (1981) Repeated seeding technique

for growing large single crystals of proteins. J Mol

Biol 147, 465–469.

109 Michel H (2012) Crystallization of membrane proteins.

In International Tables for Crystallography (Arnold E,

Himmel D & Rossmann M, eds), pp. 122–128. John

Wiley and Sons, Chicheter.

110 McPherson A, Koszelak S, Axelrod H, Day J,

Williams R, McGrath M, Robinson L & Cascio D

(1986) An experiment regarding crystallization of

soluble proteins in the presence of b-octyl-glucoside.J Biol Chem 261, 1969–1975.

111 Dock A-C, Lorber B, Moras D, Pixa G, Thierry J-C

& Gieg�e R (1984) Crystallization of transfer

ribonucleic acids. Biochimie 66, 179–201.

112 Carter CW & Carter CW Jr (1979) Protein

crystallization using incomplete factorial experiments.

J Biol Chem 254, 12219–12223.

113 Carter CW Jr, Baldwin ET & Frick L (1988)

Statistical design of experiments for protein crystal

growth and the use of a precrystallization assay.


114 Cox MJ & Weber PC (1987) Experiments with

automated protein crystallization. J Appl Cryst 20,

366–373.

115 Anderson WF, Boodhoo A & Mol CF (1988) The

importance of purity in the crystallization of DNA

binding immunoglobulin Fab fragments. J Cryst

Growth 90, 153–159.

116 Arnold E, Himmel DM & Rossmann M (2012)

Crystallography of biological macromolecules. In

International Tables for Crystallography, 2nd edn.

John Wiley and Sons, Chichester.

117 Chayen NE, Boggon TJ, Cassetta A, Deacon A,

Gleichmann T, Habash J, Harrop SJ, Helliwell JR,

Nieh YP, Peterson MR et al. (1996) Trends and

challenges in experimental macromolecular

crystallography. Q Rev Biophys 29, 227–278.

118 Wiencek JM (1999) New strategies for protein crystal

growth. Annu Rev Biomed Eng 1, 505–534.

119 Chernov AA (1993) Present-day understanding of

crystal growth from aqueous solutions. Prog Crystal

Growth Charact Mat 26, 121–151.

120 Ri�es-Kautt M & Ducruix A (1991) Crystallization of

basic proteins by ion pairing. J Cryst Growth 110,

20–25.

121 Retailleau P, Ri�es-Kautt M & Ducruix A (1997) No

salting-in of lysozyme chloride observed at low ionic

strength over a large range of pH. Biophys J 73,

2156–2163.

122 Kadri A, Damak M, Lorber B, Gieg�e R & Jenner G

(2003) Pressure vs pH phase diagrams of two

lysozymes crystallized in agarose gels. High Press Res

23, 485–491.

123 Zhang Y & Cremer PS (2009) The inverse and direct

Hofmeister series for lysozyme. Proc Natl Acad Sci

USA 106, 15249–15253.

124 Carbonnaux C, Ri�es-Kautt M & Ducruix A (1995)

Relative effectiveness of various anions on the

solubility of acidic Hypoderma lineatum collagenase at

pH 7.2. Protein Sci 4, 2123–2128.

125 Ataka M, Shinzawa-Itoh K & Yoshikawa S (1992)

Phase diagram of a crystalline membrane protein,

bovine heart cytochrome c oxidase, in the salting-in

region. J Cryst Growth 122, 60–65.

126 Odhara T, Ataka M & Katsura T (1994) Phase

diagram determination to elucidate the crystal growth

of the photoreaction center from Rhodobacter

sphaeroides. Acta Cryst D 50, 639–642.

127 Saridakis EEG, Shaw Stewart PD, Lloyd LL & Blow

DM (1994) Phase diagram and dilution experiments in

the crystallization of carboxypeptidase G2. Acta Cryst

D 50, 293–297.

128 Sauter C, Lorber B, Kern D, Cavarelli J, Moras D &

Gieg�e R (1999) Crystallogenesis studies on aspartyl-

tRNA synthetase: use of phase diagram to improve

crystal quality. Acta Cryst D 55, 149–156.

129 Lorber B & Witz J (2008) An investigation of the

crystallogenesis of an icosahedral RNA plant virus

with solubility phase diagrams. Cryst Growth Des 8,

1522–1529.

130 Schellenberger P, Demangeat G, Lemaire O,

Ritzenthaler C, Bergdoll M, Olieric V, Sauter C &

Lorber B (2011) Strategies for the crystallization of



viruses: using phase diagrams and gels to produce 3D

crystals of Grapevine fanleaf virus. J Struct Biol 174,

344–351.

131 Christopher GK, Phipps AG & Gray RJ (1998)

Temperature-dependent solubility of selected proteins.


132 Guilloteau JP, Rieskautt MM & Ducruix AF (1992)

Variation of lysozyme solubility as a function of

temperature in the presence of organic and inorganic

salts. J Cryst Growth 122, 223–230.

133 Asherie N, Ginsberg C, Blass S, Greenbaum A &

Knafo S (2008) Solubility of thaumatin. Cryst Growth

Des 8, 1815–1817.

134 Ng JD, Kuznetsov YG, Malkin AJ, Keith G, Gieg�e R

& McPherson A (1997) Visualization of RNA crystal

growth by atomic force microscopy. Nucleic Acids Res

25, 2582–2588.

135 Ri�es-Kautt M & Ducruix A (1997) Interferences

drawn from physico-chemical studies of

crystallogenesis and precrystalline state. Methods

Enzymol 276, 23–59.

136 Annunziata O, Asherie N, Lomakin A, Pande J, Ogun

O & Benedek GB (2002) Effect of polyethylene glycol

on the liquid–liquid phase transition in aqueous

protein solutions. Proc Natl Acad Sci USA 99,

14165–14170.

137 Vivar�es D, Belloni L, Tardieu A & Bonnet�e F (2002)

Catching the PEG-induced attractive interaction

between proteins. Eur Phys J E 9, 15–25.

138 Vivar�es D & Bonnet�e F (2004) Liquid–liquid phase

separations in urate oxidase/PEG mixtures:

characterization and implications for protein

crystallization. J Phys Chem B 108, 6498–6507.

139 George A & Wilson WW (1994) Predicting protein

crystallization from a dilute-solution property.

Acta Cryst D 50, 361–365.

140 Bonnet�e F, Finet S & Tardieu A (1999) Second virial

coefficient: variations with lysozyme crystallization

conditions. J Cryst Growth 196, 403–414.

141 Haas C, Drenth J & Wilson WW (1999) Relation

between the solubility of proteins in aqueous solutions

and the second virial coefficient of the solution.

J Phys Chem B 103, 2808–2811.

142 Jia YW & Liu XY (2005) Prediction of protein

crystallization based on interfacial and diffusion

kinetics. Applied Phys Lett 87, 103902.

143 Rosenberger F, Vekilov PG, Muschol M & Thomas

BR (1996) Nucleation and crystallization of globular

proteins – what do we know and what is missing.


144 Nanev CN & Tsekova D (2000) Heterogeneous

nucleation of hen-egg-white lysozyme – molecular

approach. Cryst Res Technol 35, 189–195.

145 Galkin O & Vekilov PG (2000) Control of protein

crystal nucleation around the metastable liquid–liquid

phase boundary. Proc Natl Acad Sci USA 97,

6277–6281.

146 Penkova A, Chayen N, Saridakis E & Nanev CN

(2002) Nucleation of protein crystals in a wide

continuous supersaturation gradient. Acta Cryst D 58,

1606–1610.

147 Vekilov PG (2010) Nucleation. Cryst Growth Des 10,

5007–5019.

148 Erdemir D, Lee AY & Myerson AS (2009) Nucleation

of crystals from solution: classical and two-step

models. Acc Chem Res 42, 621–629.

149 Yau S-T & Vekilov PG (2000) Quasi-planar nucleus

structure in apoferritin crystallization. Nature 406,

494–497.

150 Saridakis E, Khurshid S, Govada L, Phan Q, Hawkins

D, Crichlow GV, Lolis E, Reddy SM & Chayen NE

(2011) Protein crystallization facilitated by molecularly

imprinted polymers. Proc Nat Acad Sci USA 108,

11081–11086.

151 Georgieva DG, Kuil ME, Oosterkamp TH,

Zandbergen HW & Abrahams JP (2007)

Heterogeneous nucleation of three-dimensional protein

nanocrystals. Acta Cryst D 63, 564–570.

152 Blow DM, Chayen NE, Lloyd LF & Saridakis E

(1994) Control of nucleation of protein crystals.

Protein Sci 3, 1638–1643.

153 Bolanos-Garcia VM (2005) The tight control of

nucleation in counterdiffusive flow regimes by

hyperfluorinated oils. J Cryst Growth 283, 215–221.

154 Hammadi Z, Astier JP, Morin R & Veesler S (2009)

Spatial and temporal control of nucleation by

localized DC electric field. Cryst Growth Des 9,

3346–3347.

155 Koizumi H, Fujiwara K & Uda S (2010) Role of the

electric double layer in controlling the nucleation rate

for tetragonal hen egg white lysozyme crystals by

application of an external electric field. Cryst Growth

Des 10, 2591–2595.

156 Han Q & Lin S-X (1996) A microcrystal selection

technique in protein crystallization. J Cryst Growth

168, 181–184.

157 Chernov AA (1984) . Modern Crystallography, III

Crystal Growth Vol. 36. Springer, Berlin.

158 DeMattei RC & Feigelson RS (1992) Controlling

nucleation in protein solutions. J Cryst Growth 122,

21–30.

159 Kakinouchi K, Adachi H, Matsumura H, Inoue T,

Murakami S, Mori Y, Koga Y, Takano K &

Kanaya S (2006) Effect of ultrasonic irradiation on

protein crystallization. J Cryst Growth 292,

437–440.

160 Malkin AJ, Kuznetsov YG, Land TA, DeYoreo JJ

& McPherson A (1995) Mechanisms of growth for

protein and virus crystals. Nat Struct Biol 2,

956–959.



161 McPherson A, Malkin AJ, Kuznetsov YG, Koszelak

S, Wells M, Jenkins G, Howard J & Lawson G (1999)

The effects of microgravity on protein crystallization:

evidence for concentration gradients around growing

crystals. J Cryst Growth 196, 572–586.

162 Nakada T, Sazaki G, Miyashita S, Durbin SD &

Komatsu H (1999) Direct AFM observations of

impurity effects on a lysozyme crystal. J Cryst Growth

196, 503–510.

163 Komatsu H, Miyashita S & Suzuki Y (1993)

Interferometric observation of the interfacial

concentration gradient layers around a lysozyme

crystal. Jpn J Appl Phys 32, L1855–L1857.

164 Vekilov PG, Ataka M & Katsura T (1993) Laser

Michelson interferometry investigation of protein

crystal growth. J Cryst Growth 130, 317–320.

165 Kuznetsov YG, Malkin AJ, Greenwood A &

McPherson A (1995) Interferometric studies of

growth kinetics and surface morphology in

macromolecular crystal growth: canavalin, thaumatin,

and turnip yellow mosaic virus. J Struct Biol 114,

184–196.

166 Boggon TJ, Chayen NE, Snell EH, Dong J,

Lautenschlager P, Potthast L, Siddons DP, Stojanoff

V, Gordon E, Thompson AW et al. (1998) Protein

crystal movements and fluid flows during

microgravity growth. Phil Trans R Soc Lond A 356,

1045–1061.

167 Miyashita S, Kurihara K, Hasegawa K, Sazaki G,

Nakada T, Durbin SD, Komatsu H, Fujishima M &

Koyama M (1998) Concentration field around a

growing crystal under microgravity. J Jpn Soc

Microgravity Appl 15, 577–581.

168 Ot�alora F, Garcia-Ruiz JM, Carotenuto L,

Castagnolo D, Novella ML & Chernov AA (2002)

Lysozyme crystal growth kinetics in microgravity.

Acta Cryst D 58, 1681–1689.

169 Luft JR, Albright DT, Baird JK & DeTitta GT

(1996) The rate of water equilibration in vapor-

diffusion crystallization. Dependence on the distance

from the droplet to the reservoir. Acta Cryst D 52,

1098–1106.

170 Baird JK (1999) Theory of protein crystal nucleation

and growth controlled by solvent evaporation. J Cryst

Growth 204, 553–562.

171 Martins PM, Rocha F & Damas AM (2008)

Understanding water equilibration fundamentals as a

step for rational protein crystallization. PLoS ONE 3,

e1998.

172 Streets AM & Quake SR (2010) Ostwald ripening of

clusters during protein crystallization. Phys Rev Lett

104, 178102.

173 Thiyagarajan P & Tiede DM (1994) Detergent micelle

structure and micelle-micelle interactions determined

by small-angle neutron-scattering under solution

conditions used for membrane-protein crystallization.

J Phys Chem 98, 10343–10351.

174 Tanaka S, Ataka M, Onuma K & Kubota T (2003)

Rationalization of membrane protein crystallization

with polyethylene glycol using a simple depletion

model. Biophys J 84, 3299–3306.

175 Landau EM & Rosenbusch JP (1996) Lipidic cubic

phases: a novel concept for the crystallization of

membrane proteins. Proc Natl Acad Sci USA 93,

14532–14535.

176 Luzzati V, Tardieu A, Gulik-Krzywicki T, Rivas E &

Reiss-Husson F (1968) Structure of the cubic phases

of lipid-water systems. Nature 220, 485–488.

177 Qutub Y, Reviakine I, Maxwell C, Navarro J, Landau

EM & Vekilov PG (2004) Crystallization of

transmembrane proteins in cubo: mechanisms of

crystal growth and defect formation. J Mol Biol 343,

1243–1254.

178 Caffrey M (2011) Crystallizing membrane proteins for

structure–function studies using lipidic mesophases.

Biochem Soc Trans 39, 725–732.

179 Kubicek J, Schlesinger R, Baeken C, Buldt G, Schafer

F & Labahn J (2012) Controlled in meso phase

crystallization – a method for the structural

investigation of membrane proteins. PLoS ONE 7,

e35458.

180 Judge RA, Snell EH & van der Woerd MJ (2005)

Extracting trends from two decades of microgravity

macromolecular crystallization history. Acta Cryst D

61, 763–771.

181 Snell EH, Weisgerber S, Helliwell JR, Weckert E,

H€olzer K & Schroer K (1995) Improvements in

lysozyme protein crystal perfection through

microgravity growth. Acta Cryst D 51, 1099–1102.

182 Ng JD, Lorber B, Gieg�e R, Koszelak S, Day J,

Greenwood A & McPherson A (1997) Comparative

analysis of thaumatin crystals grown on earth and in

microgravity. Acta Cryst D 53, 724–733.

183 Declercq J-P, Evrard C, Carter DC, Wright BS,

Etienne G & Parello J (1999) A crystal of a typical

EF-hand protein grown under microgravity diffracts

X-rays beyond 0.9 �A resolution. J Cryst Growth 196,

595–601.

184 Carter DC, Lim K, Ho JX, Wright BS, Twigg PD,

Miller TY, Chapman J, Keeling K, Ruble J, Vekilov

PG et al. (1999) Lower dimer impurity incorporation

may result in higher perfection of HEWL crystal

grown in lg – a case study. J Cryst Growth 196, 623–

637.

185 Ng JD (2002) Space-grown protein crystals are more

useful for structure determination. Ann N Y Acad Sci

974, 598–609.

186 Garcia-Ruiz JM & Moreno A (1994) Investigations on

protein crystal growth by the gel acupuncture method.




187 Wakayama NI, Yin DC, Harata K, Kiyoshi T,

Fujiwara M & Tanimoto Y (2006) Macromolecular

crystallization in microgravity generated by a

superconducting magnet. Ann N Y Aca Sci 1077, 184–

193.

188 Taleb M, Didierjean C, Jelsch C, Mangeot JP,

Capelle B & Aubry A (1999) Crystallization of

proteins under an external electric field. J Cryst

Growth 200, 575–582.

189 Hansen CL, Skordalakes E, Berger JM & Quake SR

(2002) A robust and scalable microfluidic metering

method that allows protein crystal growth by free

interface diffusion. Proc Natl Acad Sci USA 99,

16531–16536.

190 Sazaki G (2009) Crystal quality enhancement by

magnetic fields. Prog Biophys Mol Biol 101, 45–55.

191 Qi J, Wakayama NI & Ataka M (2001) Magnetic

suppression of convection in protein crystal growth

processes. J Cryst Growth 232, 132–137.

192 Lorber B & Gieg�e R (1996) Containerless protein

crystallization in floating drops: application to crystal

growth monitoring under reduced nucleation

conditions. J Cryst Growth 168, 204–215.

193 Squires TM & Quake SR (2005) Microfluidics: fluid

physics at the nanoliter scale. Rev Mod Phys 77,

977–1026.

194 Sauter C, Lorber B & Gieg�e R (2002) Towards atomic

resolution with crystals grown in gel: the case of

thaumatin seen at room temperature. Proteins Struct

Funct Genet 48, 146–150.

195 Saijo S, Yamada Y, Sato T, Tanaka N, Matsui T,

Sazaki G, Nakajima K & Matsuura Y (2005)

Structural consequences of hen egg-white lysozyme

orthorhombic crystal growth in a high magnetic field:v

of X-ray diffraction intensity, conformational energy

searching and quantitative analysis of B factors and

mosaicity. Acta Cryst D 61, 207–217.

196 Sauter C, Ot�alora F, Gavira JA, Vidal O, Gieg�e R &

Garcia-Ruiz JM (2001) Structure of tetragonal hen egg

white lysozyme at 0.94 �A from crystals grown by the

counter-diffusion method. Acta Cryst D 57,

1119–1126.

197 Maki S, Murai R, Yoshikawa HY, Kitatani T,

Nakata S, Kawahara H, Hasenaka H, Kobayashi A,

Okada S, Sugiyama S et al. (2008) Protein

crystallization in a 100 nl solution with new stirring

equipment. J Synchrotron Radiat 15, 269–272.

198 Grant ML & Saville DA (1995) Long-term studies on

tetragonal lysozyme crystals grown in quiescent and

forced-convection environments. J Cryst Growth 153,

42–54.

199 Penkova A, Pan WC, Hodjaoglu F & Vekilov PG

(2006) Nucleation of protein crystals under the

influence of solution shear flow. Ann N Y Acad Sci

1077, 214–231.

200 Schall CA, Arnold E & Wiencek JM (1996) Enthalpy

of crystallization of hen egg-white lysozyme. J Cryst

Growth 165, 293–298.

201 Zielenkiewicz W (2008) Towards protein

crystallization – some thermodynamic studies.

J Therm Anal Calorim 92, 105–108.

202 Vekilov PG, Feeling-Taylor AR, Yau ST & Petsev D

(2002) Solvent entropy contribution to the free energy

of protein crystallization. Acta Cryst D 58, 1611–1616.

203 Vekilov PG, Feeling-Taylor AR, Petsev DN, Galkin

O, Nagel RL & Hirsch RE (2002) Intermolecular

interactions, nucleation, and thermodynamics of

crystallization of hemoglobin C. Biophys J 83, 1147–

1156.

204 Bergeron L, Filobelo LF, Galkin O & Vekilov PG

(2003) Thermodynamics of the hydrophobicity in

crystallization of insulin. Biophys J 85,

3935–3942.

205 Derewenda ZS & Vekilov PG (2006) Entropy and

surface engineering in protein crystallization. Acta

Cryst D 62, 116–124.

206 Yeh JI & Beale SI (2007) Calorimetric approaches to

characterizing effects of additives on protein

crystallization. Cryst Growth Des 7, 2134–2139.

207 Izumi K, Sawamura S & Ataka M (1996) X-ray

topography of lysozyme crystals. J Cryst Growth 168,

106–111.

208 Stojanoff V, Snell EF, Siddons DP & Helliwell JR

(1996) An old technique with a new application: X-ray

topography of protein crystals. Synchrotron Radiat

News 9, 25–26.

209 Nave C (1998) A description of imperfections in

protein crystals. Acta Cryst D 54, 848–853.

210 Caylor CL, Dobrianov I, Lemay SG, Kimmer C,

Kriminski S, Finkelstein KD, Zipfel W, Webb WW,

Thomas BR, Chernov AA et al. (1999)

Macromolecular impurities and disorder in protein

crystals. Proteins 36, 270–281.

211 Capelle B, Epelboin Y, Hartwig J, Moraleda AB,

Otalora F & Stojanoff V (2004) Characterization of

dislocations in protein crystals by means of

synchrotron double-crystal topography. J Appl Cryst

37, 67–71.

212 Lorber B, Sauter C, Ng JD, Zhu D-W, Gieg�e R, Vidal

O, Robert M-C & Capelle B (1999) Characterization

of protein and virus crystals by quasi-planar wave X-

ray topography: a comparison between crystals grown

in solution and in agarose gel. J Cryst Growth 204,

357–368.

213 Lovelace JJ & Borgstahl GEO (2010) The use of

orthogonal projection to visualize mosaic domains

from topographic data collected on protein crystals.


214 Sawaura T, Fujii D, Shen M, Yamamoto Y, Wako

K, Kojima K & Tachibana M (2011)



Characterization of dislocations in monoclinic hen

egg-white lysozyme crystals by synchrotron

monochromatic-beam X-ray topography. J Cryst

Growth 318, 1071–1074.

215 Hasenaka H, Sugiyama S, Hirose M, Shimizu N,

Kitatani T, Takahashi Y, Adachi H, Takano K,

Murakami S, Inoue T et al. (2009) Femtosecond laser

processing of protein crystals grown in agarose gel. J

Cryst Growth 312, 73–78.

216 Dierks K, Meyer A, Einspahr H & Betzel C (2008)

Dynamic light scattering in protein crystallization

droplets: adaptations for analysis and optimization of

crystallization processes. Cryst Growth Des 8, 1628–

1634.

217 Wilson WW (2003) Light scattering as a diagnostic for

protein crystal growth – a practical approach. J Struct

Biol 142, 56–65.

218 Borgstahl GE (2007) How to use dynamic light

scattering to improve the likelihood of growing

macromolecular crystals. Methods Mol Biol 363, 109–

129.

219 Sanchez-Puig N, Sauter C, Lorber B, Gieg�e R &

Moreno A (2012) Predicting protein crystallizability

and nucleation. Protein Pept Lett 19, 725–731.

220 Kantardjieff KA & Rupp B (2004) Protein isoelectric

point as a predictor for increased crystallization

screening efficiency. Bioinformatics 20, 2162–2168.

221 Dupeux F, Rower M, Seroul G, Blot D & Marquez

JA (2011) A thermal stability assay can help to

estimate the crystallization likelihood of biological

samples. Acta Cryst D 67, 915–919.

222 Mizianty MJ & Kurgan LA (2012) CRYSpred:

accurate sequence-based protein crystallization

propensity prediction using sequence-derived structural

characteristics. Protein Pept Lett 19, 40–49.

223 Zhu DW, Garneau A, Mazumdar M, Zhou M, Xu GJ

& Lin S-X (2006) Attempts to rationalize protein

crystallization using relative crystallizability. J Struct

Biol 154, 297–302.

224 Wukovitz SW & Yeates TO (1995) Why protein

crystals favour some space-groups over others. Nat

Struct Biol 2, 1062–1067.

225 Banatao DR, Cascio D, Crowley CS, Fleissner MR,

Tienson HL & Yeates TO (2006) An approach to

crystallizing proteins by synthetic symmetrization.

Proc Natl Acad Sci USA 103, 16230–16235.

226 Zucker FH, Stewart C, Rosa JD, Kim J, Zhang L,

Xiao L, Ross J, Napuli AJ, Mueller N, Castaneda LJ

et al. (2010) Prediction of protein crystallization

outcome using a hybrid method. J Struct Biol 171, 64–

73.

227 Dale GE, Oefner C & D’Arcy A (2003) The protein as

a variable in protein crystallization. J Struct Biol 142,

88–97.

228 Dock-Bregeon A-C, Moras D & Gieg�e R (1999)

Nucleic acids and their complexes. In Crystallization

of Nucleic Acids and Proteins A Practical Approach,

2nd edn. (Ducruix A & Gieg�e R, eds), pp. 209–243.

IRL Press, Oxford.

229 Banci L, Bertini I, Cusack S, de Jong RN, Heinemann

U, Jones EY, Kozielski F, Maskos K, Messerschmidt

A, Owens R et al. (2006) First steps towards effective

methods in exploiting high-throughput technologies

for the determination of human protein structures of

high biomedical value. Acta Cryst D 62, 1208–1217.

230 Kim Y, Bigelow L, Borovilos M, Dementieva I,

Duggan E, Eschenfeldt W, Hatzos C, Joachimiak G,

Li H, Maltseva N et al. (2008) High-throughput

protein purification for X-ray crystallography

and NMR. Adv Protein Chem Struct Biol 75,

85–105.

231 Hughes SH & Stock AM (2012) Preparing

recombinant proteins for X-ray crystallography. In

International Tables for Crystallography (Arnold E,

Himmel D & Rossmann M, eds), pp. 75–91. John

Wiley and Sons, Chicheter.

232 Kigawa T, Yamaguchi-Nunokawa E, Kodama K,

Matsuda T, Yabuki T, Matsuda N, Ishitani R, Nureki

O & Yokoyama S (2002) Selenomethionine

incorporation into a protein by cell-free synthesis.

J Struct Funct Genomics 2, 29–35.

233 Yokoyama S (2003) Protein expression systems for

structural genomics and proteomics. Curr Opin Chem

Biol 7, 39–43.

234 Jaehme M & Michel H (2013) Evaluation of cell-free

protein synthesis for the crystallization of membrane

proteins – a case study on a member of the glutamate

transporter family from Staphylothermus marinus.

FEBS J 280, 1112–1125.

235 Holbrook SR, Holbrook EL & Walukiewicz HE

(2001) Crystallization of RNA. Cell Mol Life Sci 58,

234–243.

236 Lietzke SE, Barnes CL & Kundrot CE (1995)

Crystallization and structure determination of RNA.

Curr Opin Struct Biol 5, 645–649.

237 Wernimont A & Edwards A (2009) In situ proteolysis

to generate crystals for structure determination: an

update. PLoS ONE 4, e5094.

238 Huang J, Gong Y, Huang D, Haire L, Liu J & Peng

Y (2012) ‘Seeding’ with protease to optimize protein

crystallization conditions in in situ proteolysis. Acta

Cryst F 68, 606–609.

239 D’Arcy A, Stihle M, Kostrewa D & Dale G (1999)

Crystal engineering: a case study using the 24 kDa

fragment of the DNA gyrase B subunit from

Escherichia coli. Acta Cryst D 55, 1623–1625.

240 Gallagher DT, Smith NN, Kim SK, Robinson H &

Reddy PT (2009) Protein crystal engineering of



YpAC-IV using a strategy of excess charge reduction.

Cryst Growth Des 9, 3570–3574.

241 McPherson A, Nguyen C, Cudney R & Larson SB

(2011) The role of small molecule additives and

chemical modification in protein crystallization. Cryst

Growth Des 11, 1469–1474.

242 Kim Y, Quartey P, Li H, Volkart L, Hatzos C, Chang

C, Nocek B, Cuff M, Osipiuk J, Tan K et al. (2008)

Large-scale evaluation of protein reductive

methylation for improving protein crystallization. Nat

Methods 5, 853–854.

243 Lawson DM, Artymiuk PJ, Yewdall SJ, Smith JMA,

Livingstone JC, Treffry A, Luzzago A, Levi S, Arosio

P, Cesarini G et al. (1991) Solving the structure of

human H ferritin by genetically engineering

intermolecular crystal contacts. Nature 349, 541–544.

244 Binz HK, Amstutz P, Kohl A, Stumpp MT, Briand C,

Forrer P, Gr€utter MG & Pluckthun A (2004) High-

affinity binders selected from designed ankyrin repeat

protein libraries. Nat Biotechnol 22, 575–582.

245 Sennhauser G & Gr€utter MG (2008) Chaperone-

assisted crystallography with DARPins. Structure 16,

1443–1453.

246 Low C, Moberg P, Quistgaard EM, Hedren M,

Guettou F, Frauenfeld J, Haneskog L & Nordlund P

(2013) High-throughput analytical gel filtration

screening of integral membrane proteins for structural

studies. Biochim Biophys Acta 1830, 3497–3508.

247 Mariuzza RA, Jankovic DL, Boulot G, Amit AG,

Saludjian P, Le Guern A, Mazie JC & Poljak RJ

(1983) Preliminary crystallographic study of the

complex between the Fab fragment of a monoclonal

anti-lysozyme antibody and its antigen. J Mol Biol

170, 1055–1058.

248 Hunte C & Michel H (2002) Crystallisation of

membrane proteins mediated by antibody fragments.

Curr Opin Struct Biol 12, 503–508.

249 Jancarik J & Kim S-H (1991) Sparse matrix sampling;

a screening method for crystallization of proteins.


250 Grimm C, Chari A, Reuter K & Fischer U (2010) A

crystallization screen based on alternative polymeric

precipitants. Acta Cryst D 66, 685–697.

251 Golden BL, Podell ER, Gooding AR & Cech TR

(1997) Crystals by design: a strategy for crystallization

of a ribozyme derived from the Tetrahymena group I

intron. J Mol Biol 270, 711–723.

252 Radaev S & Sun PD (2002) Crystallization of protein–protein complexes. J Appl Cryst 35, 674–676.

253 Newstead S, Ferrandon S & Iwata S (2008)

Rationalizing alpha-helical membrane protein

crystallization. Protein Sci 17, 466–472.

254 Parker JL & Newstead S (2012) Current trends in

alpha-helical membrane protein crystallization: an

update. Protein Sci 21, 1358–1365.

255 McPherson A & Cudney B (2006) Searching for silver

bullets: an alternative strategy for crystallizing

macromolecules. J Struct Biol 156, 387–406.

256 Hedderich T, Marcia M, Kopke J & Michel H (2011)

PICKScreens, a new database for the comparison of

crystallization screens for biological macromolecules.


257 Garlitz JA, Summers CA, Flowers RA 2nd &

Borgstahl GE (1999) Ethylammonium nitrate: a

protein crystallization reagent. Acta Cryst D 55, 2037–

2038.

258 Pusey ML, Paley MS, Turner MB & Rogers RD

(2007) Protein crystallization using room temperature

ionic liquids. Cryst Growth Des 7, 787–793.

259 Judge RA, Takahashi S, Longenecker KL, Fry EH,

Abad-Zapatero C & Chiu ML (2009) The effect of

ionic liquids on protein crystallization and

X-ray diffraction studies. Cryst Growth Des 9, 3463–

3469.

260 Chen XW, Ji YP & Wang JH (2010) Improvement on

the crystallization of lysozyme in the presence of

hydrophilic ionic liquid. Analyst 135, 2241–2248.

261 Santos SP, Bandeiras TM, Pinto AF, Teixeira M,

Carrondo MA & Romao CV (2012) Thermofluor-

based optimization strategy for the stabilization and

crystallization of Campylobacter jejuni

desulforubrerythrin. Protein Expres Purif 81, 193–200.

262 Thakur AS, Robin G, Guncar G, Saunders NF,

Newman J, Martin JL & Kobe B (2007) Improved

success of sparse matrix protein crystallization

screening with heterogeneous nucleating agents. PLoS

ONE 2, e1091.

263 Nederlof I, Hosseini R, Georgieva D, Luo JH, Li DF

& Abrahams JP (2011) A straightforward and robust

method for introducing human hair as a nucleant into

high throughput crystallization trials. Cryst Growth

Des 11, 1170–1176.

264 Anand K, Pal D & Hilgenfeld R (2002) An overview

on 2-methyl-2,4-pentanediol in crystallization and in

crystals of biological macromolecules. Acta Cryst D

58, 1722–1728.

265 Parmar AS, Gottschall PE & Muschol M (2007) Pre-

assembled clusters distort crystal nucleation kinetics in

supersaturated lysozyme solutions. Biophys Chem 129,

224–234.

266 Lohkamp B & Dobritzsch D (2008) A mixture of

fortunes: the curious determination of the structure of

Escherichia coli BL21 Gab protein. Acta Cryst D 64,

407–415.

267 Kors CA, Wallace E, Davies DR, Li L, Laible PD &

Nollert P (2009) Effects of impurities on membrane-

protein crystallization in different systems. Acta Cryst

D 65, 1062–1073.

268 Giffard M, Ferte N, Ragot F, El Hajji M, Castro B &

Bonnet�e F (2011) Urate oxidase purification by



salting-in crystallization: towards an alternative to

chromatography. PLoS ONE 6, e19013.

269 Plomp M, McPherson A & Malkin AJ (2003) Repair

of impurity-poisoned protein crystal surfaces. Proteins

50, 486–495.

270 Ferreira C, Rocha FA, Damas AM & Martins PM

(2012) Running away from thermodynamics to

promote or inhibit crystal growth. Cryst Growth Des

12, 40–43.

271 Adachi H, Takano K, Yoshimura M, Mori Y &

Sasaki T (2002) Promotion of large protein crystal

growth with stirring solution. Jpn J Appl Phys 41,

L1025–L1027.

272 Newman J, Egan D, Walter TS, Meged R, Berry I,

Ben Jelloul M, Sussman JL, Stuart DI & Perrakis A

(2005) Towards rationalization of crystallization

screening for small- to medium-sized academic

laboratories: the PACT/JCSG+ strategy. Acta Cryst D

61, 1426–1431.

273 Busso D, Stierle M, Thierry J-C & Moras D (2008)

Automated recombinant protein expression screening

in Escherichia coli. Methods Mol Biol 426, 175–186.

274 Kim Y, Babnigg G, Jedrzejczak R, Eschenfeldt WH,

Li H, Maltseva N, Hatzos-Skintges C, Gu M,

Makowska-Grzyska M, Wu R et al. (2011) High-

throughput protein purification and quality assessment

for crystallization. Methods 55, 12–28.

275 Ericsson UB, Hallberg BM, Detitta GT, Dekker N &

Nordlund P (2006) Thermofluor-based high-

throughput stability optimization of proteins for

structural studies. Anal Biochem 357, 289–298.

276 Vallotton P, Sun CM, Lovell D, Fazio VJ & Newman

J (2010) DroplIT, an improved image analysis method

for droplet identification in high-throughput

crystallization trials. J Appl Cryst 43, 1548–1552.

277 Khurshid S, Haire LF & Chayen NE (2010)

Automated seeding for the optimization of crystal

quality. J Appl Cryst 43, 752–756.

278 Chayen NE (2007) Optimization techniques for

automation and high throughput. Methods Mol Biol

363, 175–190.

279 Sugahara M, Shimizu K, Asada Y, Fukunishi H,

Kodera H, Fujii T, Osada E, Kasazaki T, Sawada T,

Chikusa H et al. (2010) Autolabo: an automated

system for ligand-soaking experiments with protein

crystals. J Appl Cryst 43, 940–944.

280 Viola R, Carman P, Walsh J, Miller E, Benning M,

Frankel D, McPherson A, Cudney B & Rupp B (2007)

Operator-assisted harvesting of protein crystals using a

universal micromanipulation robot. J Appl Cryst 40,

539–545.

281 Makino M, Wada I, Mizuno N, Hirata K, Shimizu N,

Hikima T, Yamamoto M & Kumasaka T (2012) Fine-

needle capillary mounting for protein microcrystals. J

Appl Cryst 45, 785–788.

282 Bingel-Erlenmeyer R, Olieric V, Grimshaw JPA,

Gabadinho J, Wang X, Ebner SG, Isenegger A,

Schneider R, Schneider J, Glettig W et al. (2011) SLS

crystallization platform at beamline X06DA – a fully

automated pipeline enabling in situ X-ray diffraction

screening. Cryst Growth Des 11, 916–923.

283 Page R & Stevens RC (2004) Crystallization data

mining in structural genomics: using positive and

negative results to optimize protein crystallization

screens. Methods 34, 373–389.

284 Newman J, Bolton EE, Muller-Dieckmann J, Fazio VJ,

Gallagher DT, Lovell D, Luft JR, Peat TS, Ratcliffe D,

Sayle RA et al. (2012) On the need for an international

effort to capture, share and use crystallization screening

data. Acta Cryst F 68, 253–258.

285 Mueller U, Nyarsik L, Horn M, Rauth H, Przewieslik

T, Saenger W, Lehrach H & Eickhoff H (2001)

Development of a technology for automation and

miniaturization of protein crystallization. J Biotech 85,

7–14.

286 Ng JD, Stevens RC & Kuhn P (2008) Protein

crystallization in restricted geometry: advancing old

ideas for modern times in structural proteomics.

Methods Mol Biol 426, 363–376.

287 Kuil ME, Bodenstaff ER, Hoedemaker FJ &

Abrahams JP (2002) Protein nano-crystallogenesis.

Enz & Microb Technol 30, 262–265.

288 Schneider TR (2008) Synchrotron radiation:

micrometer-sized X-ray beams as fine tools for

macromolecular crystallography. HFSP J 2, 302–306.

289 Hansen CL, Sommer MO & Quake SR (2004)

Systematic investigation of protein phase behavior

with a microfluidic formulator. Proc Natl Acad Sci

USA 101, 14431–14436.

290 Hansen CL, Classen S, Berger JM & Quake SR (2006)

A microfluidic device for kinetic optimization of

protein crystallization and in situ structure

determination. J Am Chem Soc 128, 3142–3143.

291 Zheng B, Roach LS & Ismagilov RF (2003) Screening

of protein crystallization conditions on a microfluidic

chip using nanoliter-size droplets. J Am Chem Soc

125, 11170–11171.

292 Yadav MK, Gerdts CJ, Sanishvili R, Smith WW,

Roach LS, Ismagilov RF, Kuhn P & Stevens RC

(2005) In situ data collection and structure refinement

from microcapillary protein crystallization. J Appl

Cryst 38, 900–905.

293 Shim JU, Cristobal G, Link DR, Thorsen T, Jia Y,

Piattelli K & Fraden S (2007) Control and

measurement of the phase behavior of aqueous

solutions using microfluidics. J Am Chem Soc 129,

8825–8835.

294 Selimovic S, Jia Y & Fraden S (2009) Measuring the

nucleation rate of lysozyme using microfluidics. Cryst

Growth Des 9, 1806–1810.



295 Selimovic S, Gobeaux F & Fraden S (2010) Mapping

and manipulating temperature-concentration phase

diagrams using microfluidics. Lab Chip 10, 1696.

296 Ng JD, Clark PJ, Stevens RC & Kuhn P (2008) In situ

X-ray analysis of protein crystals in low-birefringent

and X-ray transmissive plastic microchannels. Acta

Cryst D 64, 189–197.

297 Dhouib K, Khan Malek C, Pfleging W, Gauthier-

Manuel B, Duffait R, Thuillier G, Ferrigno R,

Jacquamet L, Ohana J, Ferrer JL et al. (2009)

Microfluidic chips for the crystallization of

biomacromolecules by counter-diffusion and on-chip

crystal X-ray analysis. Lab Chip 9, 1412–1421.

298 Gerdts CJ, Elliott M, Lovell S, Mixon MB, Napuli

AJ, Staker BL, Nollert P & Stewart L (2008) The

plug-based nanovolume Microcapillary Protein

Crystallization System (MPCS). Acta Cryst D 64,

1116–1122.

299 Yu Y, Wang X, Oberthur D, Meyer A, Perbandt M,

Duan L & Kang Q (2012) Design and application of a

microfluidic device for protein crystallization using an

evaporation-based crystallization technique. J Appl

Cryst 45, 53–60.

300 Stura E (1999) Seeding techniques. In Crystallization

of Nucleic Acids and Proteins: A Practical Approach,

2nd edn. (Ducruix A & Gieg�e R, eds), pp. 177–208.

IRL Press, Oxford.

301 Bergfors T (2003) Seeds to crystals. J Struct Biol 142,

66–76.

302 Georgiev A, Vorobiev S, Edstrom W, Song T, Laine

A, Hunt J & Allen P (2006) Automated streak-seeding

with micromachined silicon tools. Acta Cryst D 62,

1039–1045.

303 Ireton GC & Stoddard BL (2004) Microseed matrix

screening to improve crystals of yeast cytosine

deaminase. Acta Cryst D 60, 601–605.

304 D’Arcy A, Villard F & Marsh M (2007) An

automated microseed matrix-screening method for

protein crystallization. Acta Cryst D 63, 550–554.

305 Walter TS, Mancini EJ, Kadlec J, Graham SC,

Assenberg R, Ren J, Sainsbury S, Owens RJ, Stuart

DI, Grimes JM et al. (2008) Semi-automated

microseeding of nanolitre crystallization experiments.

Acta Cryst F 64, 14–18.

306 Yoshikawa HY, Hosokawa Y, Murai R, Sazaki G,

Kitatani T, Adachi H, Inoue T, Matsumura H,

Takano K, Murakami S et al. (2012) Spatially precise,

soft microseeding of single protein crystals by

femtosecond laser ablation. Cryst Growth Des 12,

4334–4339.

307 DeLucas LJ, Hamrick D, Cosenza L, Nagy L,

McCombs D, Bray T, Chait A, Stoops B, Belgovskiy

A, Wilson WW et al. (2005) Protein crystallization:

virtual screening and optimization. Prog Biophys Mol

Biol 88, 285–309.

308 Iino H, Naitow H, Nakamura Y, Nakagawa N, Agari

Y, Kanagawa M, Ebihara A, Shinkai A, Sugahara M,

Miyano M et al. (2008) Crystallization screening test

for the whole-cell project on Thermus thermophilus

HB8. Acta Cryst F 64, 487–491.

309 Kuta Smatanova I, Gavira JA, Rezacova P, Vacha F

& Garcia-Ruiz JM (2006) New techniques for

membrane protein crystallization tested on

photosystem II core complex of Pisum sativum.

Photosynth Res 90, 255–259.

310 Zhang CY, Yin DC, Lu QQ, Guo YZ, Guo WH,

Wang XK, Li HS, Lu HM & Ye YJ (2008) Cycling

temperature strategy: a method to improve the

efficiency of crystallization condition screening of

proteins. Cryst Growth Des 8, 4227–4232.

311 Meged R, Dym O & Sussman JL (2008) High

throughput pH optimization of protein crystallization.


312 Budayova-Spano M, Dauvergne F, Audiffren M,

Bactivelane T & Cusack S (2007) A methodology and

an instrument for the temperature-controlled

optimization of crystal growth. Acta Cryst D 63,

339–347.

313 Crespo R, Martins PM, Gales L, Rocha F & Damas

AM (2010) Potential use of ultrasound to promote

protein crystallization. J Appl Cryst 43, 1419–1425.

314 Newman J (2006) A review of techniques for

maximizing diffraction from a protein crystal in stilla.


315 Rayment I (2002) Small-scale batch crystallization of

proteins revisited: an underutilized way to grow large

protein crystals. Structure 10, 147–151.

316 Oksanen E, Blakeley MP, Bonnet�e F, Dauvergne MT,

Dauvergne F & Budayova-Spano M (2009) Large

crystal growth by thermal control allows

combined X-ray and neutron crystallographic studies

to elucidate the protonation states in Aspergillus

flavus urate oxidase. J Royal Soc Interface 6,

S599–S610.

317 Hazemann I, Dauvergne MT, Blakeley MP, Meilleur

F, Haertlein M, Van Dorsselaer A, Mitschler A,

Myles DA & Podjarny A (2005) High-resolution

neutron protein crystallography with radically small

crystal volumes: application of perdeuteration to

human aldose reductase. Acta Cryst D 61,

1413–1417.

318 Ferr�e-D’Amar�e AR, Zhou K & Doudna JA (1998) A

general module for RNA crystallization. J Mol Biol

279, 621–631.

319 Ravindran PP, Heroux A & Ye JD (2011)

Improvement of the crystallizability and expression of

an RNA crystallization chaperone. J Biochem 150,

535–543.

320 Ke A & Doudna JA (2004) Crystallization of RNA

and RNA–protein complexes. Methods 34, 408–414.



321 Ferr�e-D’Amar�e AR (2010) Use of the spliceosomal

protein U1A to facilitate crystallization and structure

determination of complex RNAs. Methods 52, 159–167.

322 Ye JD, Tereshk V, Frederiksen JK, Koide A,

Fellouse FA, Sidhu SS, Koide S, Kossiakoff AA &

Piccirilli JA (2007) Chaperone-assisted RNA

crystallography: selection and co-crystallization of a

Fab-Delta C209 P4-P6 complex. J Biomol Struct Dyn

24, 688–689.

323 Oubridge C, Ito N, Teo C-H, Fearnley I & Nagai K

(1995) Crystallization of RNA–protein complexes. II.

The application of protein engineering for

crystallization of the UIA protein–RNA complex. J

Mol Biol 249, 409–423.

324 Gieg�e R, Touz�e E, Lorber B, Th�eobald-Dietrich A &

Sauter C (2008) Crystallogenesis trends of free and

liganded aminoacyl-tRNA synthetases. Cryst Growth

Des 8, 4297–4306.

325 Martins P, P€essoa J, Sarkany Z, Rocha F & Damas A

(2008) Rationalizing protein crystallization screenings

through water equilibration theory and protein

solubility data. Cryst Growth Des 8, 4233–4243.

326 Itoh Y, Brocker MJ, Sekine S, Hammond G, Suetsugu

S, S€oll D & Yokoyama S (2013) Decameric

SelA*tRNASec ring structure reveals mechanism of

bacterial selenocysteine formation. Science 340, 75–78.

327 Grimm C, Klebe G, Ficner R & Reuter K (2000)

Screening orthologs as an important variable in

crystallization: preliminary X-ray diffraction studies of

the tRNA-modifying enzyme S-adenosyl-methionine:

tRNA ribosyl transferase isomerase. Acta Cryst D 56,

484–488.

328 Poodt PWG, Heijna MCR, Christianen PCM, van

Enckevort WJP, de Grip WJ, Tsukamoto K, Maan JC

& Vlieg E (2006) Using gradient magnetic fields to

suppress convection during crystal growth. Cryst

Growth Des 6, 2275–2280.

329 Espinoza-Montero PI, Moreno-Narvaez ME,

Frontana-Uribe BA, Stojanoff V & Moreno A (2013)

Investigations on the use of graphite electrodes using a

Hull-type growth cell for ellectrochemically assisted

protein crystallization. Cryst Growth Des 13, 590–598.

330 Larson SB, Day JS, Nguyen C, Cudney R &

McPherson A (2008) Progress in the development of

an alternatuve approach to macromolecule

crystallization. Cryst Growth Des 8, 3038–3052.

331 Numata T, Ikeuchi Y, Fukai S, Adachi H,

Matsumura H, Takano K, Murakami S, Inoue T,

Mori Y, Sasaki T et al. (2006) Crystallization and

preliminary X-ray analysis of the tRNA thiolation

enzyme MnmA from Escherichia coli complexed with

tRNAGlu. Acta Cryst F 62, 368–371.

332 Li L, Mustafi D, Fu Q, Tereshko V, Chen DL, Tice

JD & Ismagilov RF (2006) Nanoliter microfluidic

hybrid method for simultaneous screening and

optimization validated with crystallization of

membrane proteins. Proc Natl Acad Sci USA 103,

19243–19248.

333 Sato S, Mimasu S, Sato A, Hino N, Sakamoto K,

Umehara T & Yokoyama S (2011) Crystallographic

study of a site-specifically cross-linked protein complex

with a genetically incorporated photoreactive amino

acid. Biochemistry 50, 250–257.

334 Yoshikawa S, Kukimoto-Niino M, Parker L, Handa

N, Terada T, Fujimoto T, Terazawa Y, Wakiyama M,

Sato M, Sano S et al. (2013) Structural basis for the

altered drug sensitivities of non-small cell lung cancer-

associated mutants of human epidermal growth factor

receptor. Oncogene 32, 27–38.

335 Acosta F, Eid D, Marin-Garcia L, Frontana-Uribe

BA & Moreno A (2007) From cytochrome c crystals

to a solid-state electron-transfer device. Cryst Growth

Des 7, 2187–2191.

336 Judge RA, Johns MR & White ET (1995) Protein

purification by bulk crystallization: the recovery of

ovalbumin. Biotech and Bioengin 48, 316–323.

337 Brader ML, Sukumar M, Pekar AH, McClellan DS,

Chance RE, Flora DB, Cox AL, Irwin L & Myers SR

(2002) Hybrid insulin cocrystals for controlled release

delivery. Nat Biotechnol 20, 800–804.

338 Yonath A (2010) Polar bears, antibiotics, and the

evolving ribosome (Nobel Lecture). Angew Chem Int

Ed Engl 49, 4341–4354.

339 Koopmann R, Cupelli K, Redecke L, Nass K,

Deponte DP, White TA, Stellato F, Rehders D, Liang

M, Andreasson J, et al. (2012) In vivo protein

crystallization opens new routes in structural biology.

Nat Methods 9, 259–262.

340 Domike KR&Donald AM (2009) Kinetics of spherulite

formation and growth: salt and protein concentration

dependence on proteins beta-lactoglobulin and insulin.

Int J Biol Macromol 44, 301–310.

341 Martins PM (2013) True and apparent inhibition of

amyloid fibril formation. Prion 7, 136–139.

342 Kam M, Perl-Treves D, Sfez R & Addadi L (1994)

Specificity in the recognition of crystals by antibodies.

J Mol Recognit 7, 257–264.

343 Heijna MCR, Theelen MJ, van Enckevort WJP &

Vlieg E (2007) Spherulitic growth of hen egg-white

lysozyme crystals. J Phys Chem B 111, 1567–1573.

344 Kulkarni CV (2012) Lipid crystallization: from self-

assembly to hierarchical and biological ordering.

Nanoscale 4, 5779–5791.

345 Perederina A & Krasilnikov AS (2012) Crystallization

of RNA–protein complexes: from synthesis and

purification of individual components to crystals.


346 Mesters JR & Hilgenfeld R (2007) Protein

glycosylation, sweet to crystal growth? Cryst Growth

Des 7, 2251–2253.



347 Sugiyama S, Shimizu N, Sazaki G, Hirose M,

Takahashi Y, Maruyama M, Matsumura H, Adachi

H, Takano K, Murakami S et al. (2013) A novel

approach for protein crystallization by a synthetic

hydrogel with thermoreversible gelation polymer.


348 Gangloff M, Moreno A & Gay NJ (2013) Liesegang-

like patterns of Toll crystals grown in gel. J Appl

Crystallogr 46, 337–345.

349 Shah UV, Allenby MC, Williams DR & Heng JYY

(2012) Crystallization of proteins at ultralow

supersaturations using novel three-dimensional

nanotemplates. Cryst Growth Des 12, 1772–1777.

350 Villasenor AG, Wong A, Shao A, Garg A, Donohue

TJ, Kuglstatter A & Harris SF (2012) Nanolitre-scale

crystallization using acoustic liquid-transfer

technology. Acta Cryst D 68, 893–900.

351 Bonnefond L, Schellenberger P, Basquin J, Demangeat

G, Ritzenthaler C, Chenevert R, Balg C, Frugier M,

Rudinger-Thirion J, Gieg�e R et al. (2011) Exploiting

protein engineering and crystal polymorphism for

successful X-ray structure determination. Cryst Growth

Des 11, 4334–4343.

352 Minton AP (2001) The influence of macromolecular

crowding and macromolecular confinement on

biochemical reactions in physiological media. J Biol

Chem 276, 10577–10580.

Supporting information

Additional supporting information may be found in

the online version of this article at the publisher’s web

site:Data S1 to S10. Complete bibliography for Table 1–8

and Figs 1–4 : references [353–488] are supplemental.

Data S11. Contains additional bibliography and com-

ments on ‘Books, historical accounts & reviews on

crystal growth’, on ‘ICCBM proceedings’, and on

‘Specialized reviews & research articles’.



a historical perspective on protein crystallization from 1840 to the present day

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