Reflections on the development of

biocrystallography

in Grenoble

Stephen Cusack

European Molecular Biology Laboratory

Grenoble

Personal

Bragg Symposium

Crystallography for Life

November 28th 2013

1.2 Å

resolution

Bragg reflections

from a protein crystal

Ewald Sphere

Construction (1913)

16/12/2013 3

Solve phase problem

European

Photon and Neutron

Campus

(EPN Campus)

Integrating

Structural Biology

on the EPN Campus

European Molecular Biology

Laboratory,

Grenoble Outstation

European Synchrotron

Radiation Facility

Institute Laue-Langevin

Institute for Structural

Biology

The Partnership for

Structural Biology

since 2002

First neutrons (1971) then X-rays (1994).

ESRF(1994) EMBL(1976) ILL (1971) IBS(1990)

Yearly growth in PDB deposited X-ray structures

16/12/2013 7

2000: 11394 1990: 486

1994 ESRF opens

1971:

PDB

starts

‘Cottage industry’ ‘Mass production’

Period covered by talk

1976: 13

1976 EMBL-GR opens

ILL opens

2013:

84553

structures

Origin of Institut Laue Langevin

16/12/2013 8

1967: The ILL is founded on January 19th by France and Germany

1968: Landscaping of site

1969: Start of work on the reactor floor and walls

1971: Construction complete: reactor goes critical on Aug. 31st, ramping to full power in December.

1972: First experiments.

1973: The UK becomes an Associate on January 1st

1976: The European Molecular Biology Laboratory Outstation is established on the site

Newly constructed ILL

From mentor to student;

From the Braggs to EMBL

16/12/2013 9

Joseph John

Thomson

1897 Discovered

the electron.

Sir William

Henry Bragg

1915 Nobel

Prize

J. Desmond Bernal

1924 Graphite structure

(Royal Institution)

1927-1937

Crystallography at

Cavendish, Cambridge.

Worked on proteins in

the 1930s. M. Perutz a

student of his.

1937 Professor of

Physics at Birkbeck

College. Worked with A.

Klug, R. Franklin.

William Laurence

Bragg

1912 Inspired by Von

Laue's, introduced

Bragg's Law

1915 Nobel Prize.

1938 Moves from

Manchester to

Cambridge

Max Perutz

1936 arrives in

Cavendish Lab,

Cambridge.Starts to

work on haemoglobin

1947 MRC Unit for

Research on the

Molecular Structure

of Biological Systems

1953 Isomorphous

replacement

John Kendrew

1945 Joined Max Perutz

1947 MRC Unit for

Research on the

Molecular Structure of

Biological Systems

1957 6 Å map of sperm-

whale myoglobin

1962 Nobel Prize

Structure of myoglobin: A three-dimensional

Fourier synthesis at 2 Å resolution.

KENDREW JC, DICKERSON RE, STRANDBERG BE, HART RG,

DAVIES DR, PHILLIPS DC, SHORE VC.

Nature, 1960.

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The Nobel Prize in Chemistry 1962

16/12/2013 11

The Nobel Prize in Chemistry 1962 was awarded jointly to

Max Ferdinand Perutz

John Cowdery Kendrew

"for their studies of the structures of globular proteins"

Same year: Nobel Prize for physiology or medicine for

Jim Watson, Francis Crick and Maurice Wilkins for DNA structure

Origin of EMBL / EMBL Grenoble • 1976 - An agreement is signed establishing a second outstation at the site of the Institut Laue-Langevin

in Grenoble.

• 1975 - Construction of the Heidelberg facility begins. EMBL outstation at the DESY synchrotron ring in

Hamburg established

• 1974 - EMBL becomes a legal entity. Sir John Kendrew is appointed as its first Director General. 40th

Anniversary in 2014.

• 1971 - Heidelberg is chosen as the site for EMBL's main laboratory.

• 1969 - The first proposals to include Outstations and stronger emphasis on technological development

and service functions for the European Molecular Biology Laboratory (EMBL) are made.

• 1968 - The European Molecular Biology Conference is founded, associating

14 governments with EMBO.

• 1963 - Scientists at a meeting in Ravello, Italy form the European Molecular Biology

Organization (EMBO) to pursue the idea of the laboratory.

• 1962 - Leó Szilárd, Victor F. Weisskopf, James D. Watson and John Kendrew

meet in Geneva to discuss possibility of establishing an international laboratory

for molecular biology.

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Why an EMBL Outstation in Grenoble ?

Myoglobin crystallography: meeting point of

X-rays and neutrons

Binding of xenon to sperm whale myoglobin. Schoenborn BP, Watson HC, Kendrew JC. Nature 1965.

Neutron diffraction analysis of myoglobin. Schoenborn BP. Nature 1969.

16/12/2013 13

1971-1975 Discussions with the ILL and CENG about

setting up EMBL Outstation associated with the ILL.

Key people: John Kendrew (EMBL), Benno Schoenborn (BNL),

Rudolf Mössbauer (ILL), Jules Horowitz (CEA),

Bernard Jacrot (ILL)

1976 EMBL Grenoble Outstation opened, housed

in the Laboratoire de Moyenne Activité (CEA) and headed

by Andrew Miller from Oxford.

Benno Schoenborn

Brookhaven

National Laboratory

Grenoble Outstation 16/12/2013 14

Laboratoire de

Moyenne

Activity (LMA) Initial location

of EMBL Grenoble

Special gate

in security fence

to access ILL

EMBL Grenoble Outstation 1978 in front of LMA Andrew Miller

Sir John

Kendrew

Sir John Randall

PhD student of W.L. Bragg,

cavity magnetron for radar.

Randall Division of Cell and

Molecular Biophysics, King’s College

Stephen Cusack

Paradoxically, the Grenoble Outstation of EMBL

did not initially do protein crystallography

Main focuses:

• Collagen structure under Andrew Miller (Founding Head

of Outstation, 1976-1981)

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• Virus structure by SANS under Bernard Jacrot (First ILL

French Director, Second Head of Outstation,1981-1989)

Small plant viruses

Adenovirus

Influenza Virus

Collagen

Molecular packing in collagen

Miller A, Wray JS.

Nature, 1971 Apr 16;230.

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rat tail tendon collagen

Rat tails Low angle diffraction

from collagen fibres

(640 Å spacing)

Influenza virus structure by SANS (1985)

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Small angle neutron

scattering curves

at different H2O/D2O

ratios (contrast variation)

Spherical

Influenza B virions

Derived spherical shell model

of virion structure

Structure and composition of influenza virus. A small-angle neutron scattering study.

Cusack S, Ruigrok RW, Krygsman PC, Mellema JE. J Mol Biol. 1985.

The switch to protein crystallography 1985: Sabbatical in Harvard to learn protein

crystallography. First synchrotron experience at CHESS, Cornell

Structure of the influenza virus haemagglutinin complexed

with its receptor, sialic acid.

Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Nature. 1988.

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1986: Return to Grenoble to work on Adenovirus Fibre structure

Structure of receptor binding proteins of adenovirus

Virus-Receptor Interaction in an

Adenovirus System

Lennart Philipson, Karl Lonberg-

Holm and Ulf Pettersson, 1968

Adenovirus fibre is the receptor

binding protein

Lennart Philipson: succeeded John Kendrew as 2nd EMBL Director General

In 1982

A significant issue of Nature

16 April 1971 Vol 230 No 5294 pp417-474

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1. Synchrotron Radiation as a Source for

X-ray Diffraction page 434

G. ROSENBAUM, K. C. HOLMES & J. WITZ

2. Molecular Packing in Collagen page 437

A. MILLER & J. S. WRAY

3. Crystallization of a Second

Adenovirus Protein (the Fibre) page 456

VIVIEN MAUTNER & H. G. PEREIRA

Grenoble Outstation 16/12/2013 22

Crystallization of a Second Adenovirus Protein (the Fibre)

NATURE VOL. 230 APRIL 16 1971

Mautner and Pereira

Problem taken up again

from 1983-1990 by:

Bernard Jacrot

Christiane Devaux

Pierre Boulanger

Peter Timmins

Stephen Cusack

Carmen Berthet

using SANS, EM, MX

Cryo-EM of fragments of full-length

adenovirus fibre crystals (M. Adrian)

Packing model based

on projected EM density

Adenovirus fibre crystals

Macro-crystals of full-length Ad2 fibre

1985

Harvard

Adenovirus fibre

crystals

using

the Xentronics area

detector

Macro-crystals of full-length

wild-type adenovirus fibre

Adenovirus fibre

crystals

Oscillation photo(Daresbury)

C2: a=134 b=78 c=540, β=93

A triple beta-spiral in the adenovirus fibre

shaft reveals a new structural motif

for a fibrous proteins. Van Raaij, M., Mitraki, A., Lavigne, G. and Cusack. S.

Nature 1999.

Model of full-length

Ad2 fibre

(22 repeats, 300Å long)

Adenovirus Receptor binding

fibre structure not

solved until 1998

Repeating

motif in shaft

Late 1980s: X-ray Protein Crystallography in

Grenoble

CEA: George Buisson, Emile Druee

- operated a CAD4 four circle diffractometer.

EMBL: Carmen Berthet, Stephen Cusack

- rotating anode X-ray generator, precession

and Arndt-Wonacott rotation cameras, film scanner

ILL/CNRS: Mogens Lehman, Eva Pebay-Peyroula, Jean Vicat,

Claudine Cohen-Addad.

- eventually purchased and ran an Enraf-Nonius

FAST detector system at the CNRS (1988- )

Neutron crystallography: Graham Bentley, Anita Bentley,

Michel Roth (low resolution); Sax Mason (high resolution)

16/12/2013 27 Had to go to LURE (Paris), EMBL/DESY (Hamburg)

or Daresbury (UK) for synchrotron beamtime.

Seryl-tRNA synthetase: the first class II aminoacyl-tRNA

synthetase structure (1986-1990)

Reuben Leberman

Michael Härtlein

Carmen Berthet

Nicolas Nassar

Hassan Belrhali

16/12/2013 29

Solve phase problem

Second

Generation

Synchrotron

Source (LURE, Daresbury, DESY)

Capillary

Room. Temp.

FILM

Heavy metals

Crystals were mounted in capillaries

and measured at room-temperature

16/12/2013 30

Now a lost art !

Searching for heavy atom derivatives with a

Precession camera (basement of EMBL)

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Collecting one reflection at a time with a CAD4

to 6 Å resolution for seryl-tRNA synthetase

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Film data collection at LURE (Paris), a 2nd

generation synchrotron

• Very little automation (except rotation camera controller)

• No computers (no autoindexing, no strategy, no digital

data)

• Room temperature data collection

• Several days and several crystals for a dataset

• Manual orientation of crystals using

morphology

• Long hours in the photographic darkroom

• Systematic book keeping essential

• Several weeks to scan films and

obtain merged dataset.

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Seryl-tRNA synthetase crystals: C2 space-group

16/12/2013 34

Crystallised by

Reuben Leberman

In the absence of

autoindexing and

strategy programmes it was

essential to fully understand

the crystal morphology and

orientate crystals accordingly

Plate form

Massif form

c-axis c-axis

Manual analysis of Patterson function to find

heavy atom sites and allow phase calculation.

16/12/2013 35

Seryl-tRNA synthetase structure emerges (1989)

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But times were changing…..

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The publication reports datasets collected with 3 different sources/detectors

• Native and 2 derivatives: Xentronics detector on rotating anode source

• Dysprosium derivative: LURE film data at Dysprosium LIII edge

• ATP+serine data: Image-plate detector at EMBL-Hamburg and DORIS

storage ring at DESY.

FisM

at

2013

38

Automated data

collection

Jules Hendrix

Arno Lentfer

The 1st on-line 2D detector

able to handle MX images.

1989: The Hendrix-Lentfer Imaging plate

sounds the death knell of film

A new dominant detector system per decade

16/12/2013 39

1990s: image-plate

80 s readout

2000s: CCDs

1 s readout

2010s: pixel detector

0.001 s readout

1988: Cryo-crystallography introduced

Cryocrystallography of biological macromolecules: a generally applicable method.

H. Hope. Acta Cryst. B.1988

Abstract …..This approach prevents freezing of the solvent in the crystals, so that they

maintain their crystallographic integrity. Significant improvement of resolution can result from

the cryogenic data collection, and radiation damage in the cooled crystals is greatly reduced,

or eliminated, for the duration of data collection.

16/12/2013 40

Cryocrystallography of ribosomal particles.

Hope H, Frolow F, von Böhlen K, Makowski I, Kratky C, Halfon Y, Danz H, Webster P,

Bartels KS, Wittmann HG, et al.

Acta Cryst. B.1989

Mounting of crystals for macromolecular crystallography in a free-standing thin film.

T.-Y. Teng. J. Appl. Cryst. (1990). Cryoloop)

Grenoble Outstation 16/12/2013 41

Homemade

cryo-cooling system

installed at EMBL

Grenoble (1989) Stephen Curry

Thomas Thuene

Stephen Curry

Celebrating Crystallography —

a short film made by the Royal

Institution (2013).

http://richannel.org/celebrating

-crystallography

Oxford cryostream

16/12/2013 42

The Oxford Cryostream Cooler was first

launched around twenty-five years ago, and

we like to think that over the intervening

years, Oxford Cryosystems has helped

revolutionise data collection at low

temperatures.

J. Cosier and A.M. Glazer. J. Appl. Cryst. (1986). A

nitrogen-gas-stream cryostat for general X-ray diffraction

studies.

The origin of the ESRF http://www.esrf.eu/about/organisation/key-dates

16/12/2013 43

1994: The ESRF opens its doors to users, offering 15 operational

beamlines.

1994: Storage ring current raised to 150 mA

1992: First electron beam in the ring and first X-ray beam in a

beamline. Commissioning phase. Design goal reached of 100 mA

current in storage ring.

1988: Construction of the European synchrotron began, with 12

countries supporting the project.

1987: Foundation phase report. Design goals: Storage ring current 100

mA; brilliance (5 m undulator) = 2x1018 photons/mm2/mrad2/0.1%bw.

1985: Grenoble chosen as the future location of the ESRF, in order to

benefit from the presence of the neighbouring ILL and outstation of the

EMBL. (Much to the disgruntlement of Strasburg !)

1975: Launch of the idea for a European synchrotron: first meeting to

consider the feasibility of producing hard X-rays with high brilliance held

under the auspices of the European Science Foundation.

16/12/2013 44

The construction of the ESRF

16/12/2013 45

The coming of the ESRF

• First 3rd generation synchrotron source in the world

• High brilliance beams

• Intensity is high enough for radiation damage to be a

problem even for cryo-cooled crystals

• Prompted major innovations in the use of microcrystals

• Initial microcrystal experiments performed on microfocus

beamline ID13 by local Grenoble MX users with Christian

Riekel and his team.

• Small is beautiful: protein microcrystallography. Cusack,

S., Belrhali, H., Bram, A., Burghammer, M., Perrakis, A.

and Riekel C. Nature Struc. Biol. Synchrotron

Supplement (1998).

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Diffraction Images from

microfocus beamline ID13

Bacteriorhodopsin microcrystals grown in

lipidic cubic phase

16/12/2013 47

X-ray structure of bacteriorhodopsin at 2.5 Å from microcrystals grown in

lipidic cubic phases.

Pebay-Peyroula E, Rummel G, Rosenbusch JP, Landau EM.

Science. 1997

First custom designed microdiffractometer for

protein crystallography (1998)

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Protein microcrystals and the design of a microdiffractometer: current experience and

plans at EMBL and ESRF/ID13. Perrakis A, Cipriani F, Castagna JC, Claustre L, Burghammer M, Riekel C, Cusack S.

Acta Cryst. D. 1999

• Designed and built by Florent Cipriani and his team (EMBL)

• Introduced high precision air bearing spindle, along beam

crystal viewing (no parallax) and automation

• Set the standard for all future MX diffractometers.

MD1 installed on ID13 in 1998

16/12/2013 49

Synchrotrons 2nd generation 3rd 4th/FEL

Detectors Film/Image-plate CCD/Pixel ??

Min. Crystal 500/50-100 5 ??

size ( m)

Mounting Capillary Cryo-loop ??

Exposure/° 1-10m 10-30s 1-5s ??

Automation None High/Remote control ??

Structure MIR MAD SAD/S-SAD ??

Solution

Software Individual programs

History Now Future

Pipelines ??

See talk by Ilme Schlichting

Protein crystals,

Protein diffraction patterns

and

protein structures

are beautiful !

Acknowledgements

16/12/2013 51

Jo Zaccai, Eva Pebay-Peyroula, Keith Wilson, Stephen Curry,

ILL archives, EMBL archives, Wikipedia

for help in preparing the presentation

Jo Zaccai and Jean-Louis Hodeau

for the invitation