cristallogenèse biologique : de la théorie à la...

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 – [email protected]@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)


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


[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


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


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


Grapevine fanleaf virus:Grapevine fanleaf virus:producing producing

high quality crystalshigh quality crystalsfor structure determinationfor structure determination


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


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???


Sample characterizationSample characterization

Dynamic light scattering

% in

tens

ity

monodisperse

diameter (nm)

% in

tens

ity

polydisperse

30diameter (nm)100 nm

100 nm

Negative staining


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)


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 %


GFLV diffracting beyond 3 GFLV diffracting beyond 3 ÅÅ resolution resolution

FIP - BM30 (ESRF)0.2° / 120 s


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)


Two viruses, two phase diagramsTwo viruses, two phase diagrams

GFLV-TD

GFLV-F13


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


Solution vs gelSolution vs gel

In usual growth conditions➔ gradient of concentration gradient of density➔ convectionconvection➔ growth defectsgrowth defects (gaps, dislocations,

impurities...)➔ sedimentationsedimentation


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)


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


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


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


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


GFLV60 subunits30240 amino acids237291 non H atoms MW = 4.600.000


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


MicrofluidicsMicrofluidicsinin

biologicalbiologicalcrystal growthcrystal growth


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


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


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)


ChipX in briefChipX in brief


Automated on chip data collectionAutomated on chip data collection

MovieMovie


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

✔ ...


The µ-crystallogenesis teamThe µ-crystallogenesis team

Anne Théobald-Dietrich

Claude Sauter

Bernard Lorber

Richard Giegé

Kaouthar Dhouib


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...

Merciet à votre santé !

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