original document: developed by tristan fiedler for 2003 aca summer school in macromolecular...
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Original document:developed by Tristan Fiedler for
2003 ACA Summer School in Macromolecular Crystallography
Augmented 6 July 2004 by Andy HowardRebuilt for Biology 555, spring 2008
Protein CrystallizationTheory & Practice
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krystallos?krustallos?
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Journalist’s Criteria
WhoWhatWhenWhereWhyHow
Whither
Whence
(Wherefore)
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Who makes macromolecular crystals?
Macromolecular crystals are almost always produced artificially, i.e., by human action
So “who” is “scientists”
QuickTime™ and aTIFF (Uncompressed) decompressor
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What are macromolecular crystals?
Crystals are translationally ordered arrays of moleculesMacromolecular crystals are held together by relatively weak ionic intermolecular forcesSolvent content generally above 40%
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When have we made them?
Story goes back into the mid-19th centurySystematic search for crystallization conditions dates from the 1950’sScreening kits: concept 1980’s, commercialization in 1990’sRobotics late 1990’sNanoscale techniques early 21st century
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Very old (reproduction, genetic duplication)
Empirical (trial & error -- ‘Screens’)
‘Art’ vs ‘Science’
Crystallization
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1853 Hemoglobin Lehman, CG. Lehrbuch der physiologische
Chemie. Leipzig
1926 Urease Sumner, JB. J Biol Chem. 69: 435
1930 Pepsin & other proteolytic enzymes Northrop, JH, Kunitz, M, Herriot RM. Crystalline Enzymes. Columbia
University Press, NY (review)
1934 Pepsin Diffraction Bernal JD & Crowfoot, D. Nature,
133:794
1935 Tobacco Mosaic Virus Stanley, WM. Science. 81:
644
Short History1946 Nobel (Chemistry)
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Short history (concluded)
1946: Sumner Nobel prize
1958: Myoglobin structure
1959: Hemoglobin structure
1962: Perutz/Kendrew Nobel
1979: Carter&Carter paper
1985: first microgravity experiments
1990’s: commercial screening kits
Late 1990’s: viable commercial crystallization robots
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Where do we grow them?
Under mild laboratory conditions
Contrast to inorganic small molecules, which are often grown from a melt
Even small organic crystals often exploit temperature dependence
Proteins usually avoid these techniques…
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Inorganic - cooling a hot saturated substance
Polar organic - same or ppt from aqueous using organic solvents
Proteins - Yeoww!! (denature)
1.Dissolved in buffer + ppt [low]
2.controlled evaporation [higher]
Growing [Protein] Crystals
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Why do we grow them?
Because we want to know the macromolecule’s structure!
Fundamental postulate:The structure of a protein in a crystal differs only slightly, and then only on the surface, from that its soluble or membrane-associated (biologically active) form
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Do we believe this?
Short answer: yes
Skepticism was rampant through the 1970’s and has only gradually diminished
Various experimental demonstrations
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Evidence that(xtal) = (solution)
Enzymatic activity of crystals (1970’s)
Similarity of multiple crystal forms
Comparisons to NMR structures
Consistency with other biophysical techniques
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When is the postulate wrong?
Some external loops held in wrong positions (Interleukin-8)Much more common: crystal structure shows us one conformer; other conformers, and the transitions among them, are relevant
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How to make 3-D crystals
In general it involves creation of three-dimensional order
In practice with macromolecules that means creating conditions in which intermolecular forces can be exerted in the same way on each molecule
These intermolecular forces arePolar
Often water-mediated
Weak
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How do we grow macromolecular crystals?
Short answer: we gradually decrease the solubility of the protein in a way that produces ordered (crystalline) precipitation rather than disordered (amorphous) precipitation
Recognize the stages in crystallization
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Stages of crystallization
Nucleation
Governed by short-range intermolecular interactions
We want a few stable nuclei, not a lot!
Growth
Adding one molecule at a time to the nucleus
Incorrect additions lead to instability and…
Cessation of growth
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Know your protein
Cysteines
Substrates / Ligands
Proteolytic sensitivity
Metal binding
pH & Temp for stability / activity
Post Translational Modification
Protein Preparation
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Purify from natural sources
Create an expression construct
Add Tags to aid purification
6-His, Biotin-Strept., Calmodulin Bind. Peptide, GST, Maltose Bind. Protein
Expression systems
E.coli - no post translational modifs
Yeast - euk, may be better for secreted pro’s
Baculovirus-Insect & Mammalian cells
Protein Preparation
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Purification StrategyOptimize Protein ExpressionPreparation of soluble cell-free extractAMS/PEG fractionationAffinity Chromatography
Ion Exchange ChromatographySize Exclusion ChromatographyHomogeneity Analysis (SDS-PAGE, MS, DLS)
Protein Preparation
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Protein vs SaltProperty Salt Protein
Size cm’s < 1mmlarger often twinned
Integrity ElectrostaticCharged Ions
Hydrogen bondsHydrated Molecules
Solvent content LowerHigher
allows ligand access & activity
Fragile ? (needle test)
Less More
Keep Protein Crystals Hydratedin “Mother Liquor”
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Protein StorageOxidation, Deamination, Denaturation, Proteolysis, AggregationGeneral Rule : Store [x] & purified> 1 mg/mlReducing agents, in vivo pHKeep on ice / quick freeze in aliquotsFilter sterilize, Antimicrobial agents
Protein Preparation
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Solubility CurveBelow S - no ppt
Zone 1 - Metastablerare nucleationsustains growth (seed)
Zone 2- Nucleationcrystals grow
Zone 3 - Precipitation
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Does it always work this way?
No. Some proteins are more soluble in high salt than low.
Same general principles apply as long as we understand the dynamics
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What does aggregation do?
In a sense, a crystal is an aggregate…
The formation of oligomeric, randomly oriented aggregates is not conducive to crystallization
We’ll see a useful tool next week for detecting aggregation
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Second virial coefficient
Characterizes two-body interactions between protein molecules in dilute solution
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What do we do with that?
It can be measured through static light-scattering and SANS measurements
Good correlation with nucleation conditions, at least with favorable proteins
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Batch
Dialysis
Vapor Diffusion
Crystallization Methods
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Dialysis
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Double Dialysis
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Vapor Diffusion
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Vapor Diffusion Variants
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Physical:Temp, pressure, surface, viscosity, vibrationChemical:pH, precipitant, ionic strength, metalsBiochemical:purity, ligands, post-TL, proteolysisEngineering:solubility, fusion proteins, heavy atom sites
Factors affecting Crystallization
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Don’t be deceived!http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial1.html
Beautiful - No diffraction
Ugly - 1.6 Angstroms !
Moral : Its the diffraction that counts
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Good - nonamorphous, birefringent, redissolves
Bad - skin, does not redissolve, characteristic brownish tinge
Precipitateshttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
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http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html
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Whence came we?
It used to be really hard:Inadequate quantityInadequate purityUnsystematic approachesMacro quantities required
Motivation to improve crystallization approaches came as the field matured
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Whither?
High-throughputBetter protein purity
Higher quantities when required
Approaches that don’t require large quantities have appeared
More systematic approachesAutomation at every stage, including visualization
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Automated crystallization
Sample loading, distribution
visualization, decision-making
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QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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Can we get away with not knowing our protein?
Often, yes: cf. Structural genomics projects
Ugly cases (e.g. transmembrane): we still argue that the more you know, the more likely you are to get good crystals
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www.hamptonresearch.com
www.emeraldbiostructures.com
Protein Crystallization (ed. T. Bergfors) http://xray.bmc.uu.se/~terese/
Crystallization of Nucleic Acids and Proteins (ed. A. Ducruix & R. Giege)
http://www.hwi.buffalo.edu/High_Through/High_Through.html
International Tables for Crystallography. Vol. F
Part 3 : Techniques of Molecular Biology (S. Hughes & A. Stock)
Part 4 : Crystallization (R. Giege et al.)
References