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Cristallogenèse biologique :Cristallogenèse biologique :de la théorie à la pratiquede la théorie à la pratique
Claude SauterClaude Sauter
Architecture et Réactivité de l'ARNArchitecture et Réactivité de l'ARNInstitut de Biologie Moléculaire et CellulaireInstitut de Biologie Moléculaire et Cellulaire
IBMC – CNRS – Strasbourg – FranceIBMC – CNRS – Strasbourg – Francec.sauter@unistra.frc.sauter@unistra.fr
Atelier CRISTECH 2010Atelier CRISTECH 2010
A brief history of biocrystallography...A brief history of biocrystallography...
.
Giegé & Sauter, HFSP journal (2010)Giegé & Sauter, HFSP journal (2010)
Atelier CRISTECH 2010Atelier CRISTECH 2010
Biological crystallogenesisBiological crystallogenesis
Understanding crystal growth from a physical chemical viewpoint
Providing efficient strategies to grow crystals for structural biology
from molecules to their 3D structure
Atelier CRISTECH 2010Atelier CRISTECH 2010
[Agent cristallisant ]
[Ma
cro
mo
lécu
le]
Physical & chemical parameters Physical & chemical parameters
T, pH, P, ionic strength, supersaturation, nature of the crystallant,
influence of gravity, electric or magnetic fields, impurities,
crystallization method and geometry...
Biological Biological
variables and variables and
constraints constraints
limited quantity,
tricky production,
stability, ligands,
heterogeneity of
sequence and/or
conformation...
A multiparametric processA multiparametric process
Solubilitycurve
Undersaturated solution
Supersaturated solution
Nucleationzone
Metastablezone
B
AC
Atelier CRISTECH 2010Atelier CRISTECH 2010
30 years of crystallogenesis30 years of crystallogenesis
Moras et al, Nature (1980) Moras et al, Nature (1980) Ruff et al., Science (1991)Ruff et al., Science (1991)
Sauter et al., J. Mol. Biol. (2000Sauter et al., J. Mol. Biol. (2000))
MicrogravityMicrogravity
Importance ofImportance ofpurity and homogeneitypurity and homogeneity
Dynamic light scatteringDynamic light scattering
Phase diagramsPhase diagrams
ContainerlessContainerlesscrystallizationcrystallization
High pressureHigh pressure
Magnetic fieldsMagnetic fields
Convection free mediaConvection free media
Atelier CRISTECH 2010Atelier CRISTECH 2010
A few aspects of biocrystallogenesisA few aspects of biocrystallogenesis
Grapevine fanleaf virus story✔ Getting a crystallizable sample
✔ Defining reproducible
crystallization conditions
✔ Optimizing crystal quality
Microfluidics in biological crystal growth✔ Novel crystallogenesis tools
Atelier CRISTECH 2010Atelier CRISTECH 2010
Grapevine fanleaf virus:Grapevine fanleaf virus:producing producing
high quality crystalshigh quality crystalsfor structure determinationfor structure determination
Atelier CRISTECH 2010Atelier CRISTECH 2010
The GFLV peopleThe GFLV people
IBMP – Strasbourg (F)Pascale SchellenbergerChristophe RitzenthalerMarc Bergdoll
INRA – Colmar (F)Pascale SchellenbergerOlivier LemaireGérard Demangeat
IBMC – Strasbourg (F)Bernard Lorber
Claude Sauter
Swiss Light SourceVilligen (CH)
Vincent Oliéric
Atelier CRISTECH 2010Atelier CRISTECH 2010
The fanleaf diseaseThe fanleaf disease
healthyinfected
✔ Present worldwide (60% of French vineyard!)✔ Dramatic drop of yield and quality✔ No effective treatment✔ Icosahedral RNA virus
GFLV Grapevine fanleaf virus Nematode
Xiphinemaindex
1mm???
Atelier CRISTECH 2010Atelier CRISTECH 2010
Sample characterizationSample characterization
Dynamic light scattering
% in
tens
ity
monodisperse
diameter (nm)
% in
tens
ity
polydisperse
30diameter (nm)100 nm
100 nm
Negative staining
Atelier CRISTECH 2010Atelier CRISTECH 2010
Optimizing initial hitsOptimizing initial hits
16 mg/ml GFLV7.5% PEG 3350 (m/v)0.1 M Hepes Na pH 7.20.2 M proline
5 mg/ml GFLV8% PEG 3350 (m/v)
Atelier CRISTECH 2010Atelier CRISTECH 2010
Exploring GFLV phase diagramExploring GFLV phase diagram
Single crystal per drop / reproducible conditions
[ PEG 3350 ]
[ virus ]
1 mg/ml
10 mg/ml
1 µl sitting drops (vapor diffusion)
Solubility curve
2 %12 %
Atelier CRISTECH 2010Atelier CRISTECH 2010
GFLV diffracting beyond 3 GFLV diffracting beyond 3 ÅÅ resolution resolution
FIP - BM30 (ESRF)0.2° / 120 s
Atelier CRISTECH 2010Atelier CRISTECH 2010
Strange behavior...Strange behavior...
[ PEG 3350 ]
[ virus ]
1 mg/ml
10 mg/ml
4 mg/ml
7 %2 mg/ml
3 %
100um
100um
2 %12 %
Wild type
GFLV-F13
Transmission defective mutant G297D (GFLV-TD)
Atelier CRISTECH 2010Atelier CRISTECH 2010
Two viruses, two phase diagramsTwo viruses, two phase diagrams
GFLV-TD
GFLV-F13
Atelier CRISTECH 2010Atelier CRISTECH 2010
X06DA - PXIII (SLS)0.3° / 2 s
GFLV-F13: not so bad, but...GFLV-F13: not so bad, but...
100um
… optimizationis required!
=> crystals in agarose
Atelier CRISTECH 2010Atelier CRISTECH 2010
Solution vs gelSolution vs gel
In usual growth conditions➔ gradient of concentration gradient of density➔ convectionconvection➔ growth defectsgrowth defects (gaps, dislocations,
impurities...)➔ sedimentationsedimentation
Atelier CRISTECH 2010Atelier CRISTECH 2010
Solution vs gelSolution vs gel
In usual growth conditions➔ gradient of concentration gradient of density➔ convectionconvection➔ growth defectsgrowth defects (gaps, dislocations,
impurities...)➔ sedimentationsedimentation
In microgravity (g=0)In microgravity (g=0)
In gels, capillary tubes, microfluidic channels (l<100 µm)In gels, capillary tubes, microfluidic channels (l<100 µm)➔ no convection => pure diffusion pure diffusion ➔ no sedimentation => 3D growth 3D growth➔ higher crystal quality: mosaicity and I/sig(I)higher crystal quality: mosaicity and I/sig(I)➔ improved stability, cryocooling, soaking...improved stability, cryocooling, soaking...
Lorber et al.Lorber et al.Crystal growth of proteins, Crystal growth of proteins, nucleic acids, and viruses in gelsnucleic acids, and viruses in gelsProg. Biophys. Mol. Biol.Prog. Biophys. Mol. Biol. (2009)(2009)
Atelier CRISTECH 2010Atelier CRISTECH 2010
100um
Improving crystal quality in gelImproving crystal quality in gel
in solution
Diffraction at 5.5-8 Å
GFLV-F13
Schellenberger et al. submittedSchellenberger et al. submitted
100um
Diffraction at 3-4 Å
+ 0.2% (m/v) agaroseGFLV-F13
Atelier CRISTECH 2010Atelier CRISTECH 2010
Two viruses, two crystal formsTwo viruses, two crystal forms
wild type GFLV
a=279.4, b=279.4, c=293.3
α=102.4°, β=116,4°, γ=108,2°
GFLV-TD (G297D)
α = β = γ = 90°
a = b = c = 408
Asymmetric unit 20-mer
Resolution (Å)
Rmeas (%)
I/ σ(I)
60-mer
135 - 3.0 36 - 2.7
Nb unique reflections 1 214 336 (73 170) 563 202 (32 488)
Completeness (%) 88.1 (71.7) 91.6 (72.0)
9.2 (2.3) 18 (1.9)
14.1 ( 35.0) 11.9 (68.1)
Space group P1 P213
Schellenberger et al. submittedSchellenberger et al. submitted
Atelier CRISTECH 2010Atelier CRISTECH 2010
The Grapevine Fanleaf virusThe Grapevine Fanleaf virus
Patrick BronStefano Trapani CBS – Montpellier (F)
P213
Resolution
Rwork
Rfree
2.7Å
19%
21%
P1
3Å
19%
21%
AMoRe
Coot
Phenix
Atelier CRISTECH 2010Atelier CRISTECH 2010
GFLV-F13 GFLV-F13 vsvs GFLV-TD GFLV-TD
Gly297AspβG-βH loop
The structures are almost identicalRMSD = 0.16Å (504 Cα)
=> a single mutation abolishes the virus transmissionalters the solubility
Atelier CRISTECH 2010Atelier CRISTECH 2010
GFLV60 subunits30240 amino acids237291 non H atoms MW = 4.600.000
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What GFLV tells us...What GFLV tells us...
✔ Quality control is crucial
✔ The phase diagram: a powerful optimization tool
✔ Crystal growth in gel:✔ improves the crystal quality✔ facilitates crystal handling✔ improves cryocooling
Atelier CRISTECH 2010Atelier CRISTECH 2010
MicrofluidicsMicrofluidicsinin
biologicalbiologicalcrystal growthcrystal growth
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Microfluidic technologyMicrofluidic technology
✔ handling small volumes of solution✔ parallel processing of samples✔ small – fast – cheap✔ lab-on-a-chip concept✔ many applications to come in
biomedical diagnostic, chemistry, molecular biology...
Advantages for crystal-growth✔ small samples✔ high throughput screening✔ convection-free environment
Hansen et al. Hansen et al. PNAS (2002)PNAS (2002)
Quake groupQuake groupCaltechCaltech
Atelier CRISTECH 2010Atelier CRISTECH 2010
The CD crystallization chipThe CD crystallization chip
The ChipX project✔ crystallization chip using counter-diffusion (CD)✔ transparent material (observation, X-ray diffraction)✔ easy to use => no valve, no pump✔ simple design => low cost chip?!?✔ lab-on-a-chip concept: from solution to diffraction
Lilian JacquametJérémy OhannaJean-Luc Ferrer
Rosaria FerrignoMathieu BrunPierre Morin
Anne-Laure DemanJ.-François Château
Chantal Khan-MalekBernard Gauthier-Manuel
Gaël ThuillierMohamed Sahli
Atelier CRISTECH 2010Atelier CRISTECH 2010
On chip counter-diffusionOn chip counter-diffusion
L x l x h = 1.5 cm x 100 µm x 100 µm => L x l x h = 1.5 cm x 100 µm x 100 µm => 150 nl150 nl
Counter-Diffusion
Sauter, Dhouib & Lorber, Cryst. Growth Design (2007)Sauter, Dhouib & Lorber, Cryst. Growth Design (2007)Dhouib et al., Lab Chip (2009)Dhouib et al., Lab Chip (2009)
Atelier CRISTECH 2010Atelier CRISTECH 2010
Automated on chip data collectionAutomated on chip data collection
MovieMovie
Atelier CRISTECH 2010Atelier CRISTECH 2010
Microfluidic perspectivesMicrofluidic perspectives
✔ Convection-less nanoreactors✔ Extreme miniaturisation and
parallelisation✔ Many advantages for basic
investigations and for crystal growth in general (DLS, video)
✔ In situ crystal characterization using automated systems
✔ ...
Atelier CRISTECH 2010Atelier CRISTECH 2010
The µ-crystallogenesis teamThe µ-crystallogenesis team
Anne Théobald-Dietrich
Claude Sauter
Bernard Lorber
Richard Giegé
Kaouthar Dhouib
Atelier CRISTECH 2010Atelier CRISTECH 2010
In conclusionIn conclusion
✔ Biocrystallogenesis: a multidisciplinary field
✔ The rules are the same but biomolecules are more fragile and their crystals as well as
✔ Quantities are often limiting
✔ Dedictated miniaturized methods are required
✔ Current challenges: large complexes, molecular machines, membrane proteins...
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