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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
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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
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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
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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
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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.
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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
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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
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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
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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.
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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
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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.
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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
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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
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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.
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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
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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
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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