Crystallization Methods and Screening · 0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 10 12 14 16 18 20...
Transcript of Crystallization Methods and Screening · 0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 10 12 14 16 18 20...
Juan Manuel García-Ruiz
Laboratorio de Estudios CristalográficosCSIC-Universidad de Granada, Spain
Crystallization Methods and Screening
Crystallization methods
water removal, either by: Evaporation. Dialysis.
solubility change driven by: Temperature pH Dielectric constant Ionic strength Polymers
Because of lack of material protein crystallization techniques are microscale or nanoscale design
A. McPherson, Crystallisation of Biological Macromolecules, Cold Spring Harbor Laboratory Press, 1999.A.Ducruix and R.Giege (eds) Crystallisation of Nucleic Acids and Proteins: A Practical Approach, IRL Press at Oxford University 1999Methods in Enzimology, volume 368 (2003)Journal of Structural Biology, volume 142 (2003)Proceedings of the ICCBMs at Acta Crystallographica (2001 and 2003) and Journal of Crystal Growth (1998)
Solvent is water (plus detergent in membrane protein crystallisation)
Low reproducibilityLack of understanding
Crystallizability: the propensity of a compound to crystallize, i.e. to enter the a7rac8ve regime to make ordered arrays of their molecules, both with orienta8onal and transla8onal symmetry.
Crystallizability depends on the proper8es of the macromolecule itself and on the chemical cocktail used to bring there into the a7rac8ve regime, including ligands that are assumed to help crystallizability.
However, the way to mix the macromolecules and the molecules of the precipita8ng cocktail, the rate at which they mix and how fast supersatura8on is achieved, depends on the crystalliza,on technique.
Thus, the output from any crystallization experiment depends, in some extent, on the crystallization technique used. This is particularly true for screening
Vapour diffusion technique
Advantages
Drawbacks
• Easy to understand by the users• Simple to implement• Inexpensive• Robo8c available for H-‐T structural genomics• Crystals can be handled for cryoXtallography
• The solu8on moves from equilibrium to far from equilibrium• Ambiguous interpreta8on of drop’s crystalliza8on phenomena • Problems of reproducibility and scale-‐up• Each drop experiment screens a narrow crystalliza8on space (measured by visited values of supersatura8on and supersatura8on rate)
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Batch method
Direct mixing of protein and precipitant
Protein Precipitant
Trial is blind• Super(under)saturation is immediately achieved• Screening performed by repeating a number N of experiments using different initial conditions • Higher the number N of experiments higher the possibility of success• Number of visited crystallization conditions per trial: One supersaturation value, One (inLinite) rate of
supersaturation
Oil thin layer
Slow evaporation
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Precipitating agent concentration
Evaporation of water increases the concentration of precipitating agent and protein at the same rate.
and the concentration of protein decreases
As a result protein precipitates hopefully as crystals …
Vapour diffusionHanging/Si1ng drop
Transport of water molecules
Crystallisationin the drop
Protein solution + precipitant at low concentration
Precipitant at high concentration(usually twice the one in the drop)
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Note that the soluBon moves from equilibrium to out of equilibrium:It means iniBal condiBons and/or rate of evaporaBon need to be tuned
Experimental set-up
A Mach-‐Zehnder interferometer study
mirror 0
mirror 1
mirror 2
sample
633 nm
Laser
filter
collimator
Beam spliVer1
Beam spliV
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The drop was sandwiched between two glass plates with a gap of 0.4 mm.
Precipitant solu7on
T = 20ºC
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Low concentration =Low density solution
Dynamics of a crystallising dropA demonstration of the role of gravity
Vapour diffusionHanging/Si1ng drop
Growing crystal
Mach-Zehnder interferometry
Crystals sedimentate
Crystals nucleate at the surface of the drop
Convective flow motion
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Nucleation frequency J is defined as the number of stable nuclei forming per unit of time and unit of volumen. It is given by:
Scale-‐up problems
The most common precipitants used in macromolecular crystallization
Salts Volatile organic compounds
Polymers Non-volatile organic solvents
1. Propanol and isopropanol
2. 1,3-propanediol3. Dioxan4. Acetone5. Methanol6. Ethanol
1. Poly(ethylene glycol) 1000, 3350, 6000, 8000, 20000
2. Jeffamine3. Polyamine
1. 2-methyl-2,4-pentanediol2. Ethylene glycol 400
1. Ammonium phosphate and sulfate
2. Lithium sulfate3. Sodium or potassium or
ammonium chloride, phosphate, acetate, tartrate
4. Magnesium or calcium sulfate, chloride
5. Sodium or magnesium formate
The Yirst step of a Protein Crystallization experiment is always mixing The way to mix the macromolecules and the molecules of the precipitating cocktail, the rate at which they mix and how fast supersaturation is achieved, depends on the crystallization technique.
The chemical cocktail is in most cases Protein + water +
buffer + precipitant
Problem: Low reproducibility; Same chemistry yields different results within a single drop withing chemically identical drops: Lack of understanding
Precipitant
Protein
Density of protein soluBon = density precipitant soluBon
Protein heavier
Protein lighter
RelaBve viscosiBes also mater
¿Precipitant over protein or protein over precipitant?
Problems of Reproducibility
Case 1: An interferometric video corresponding to the experiment of pouring a lighter NaCl solution (1%) over a denser Lysozyme solution (30 mg/ml). The diffusivity of the protein molecules towards the upper salt solution is slower than the diffusivity of the salt molecules towards the lower protein solution. This triggers a convective mechanism that enhances mixing. Time scale, 2 frame per second.
Case 2: An interferometric video corresponding to the experiment consisting of pouring a NaCl solution (2%) over a lighter Lysozyme solution (30 mg/ml). Notice that the system becomes turbulent as a result of the development of Rayleigh-‐Taylor instability and the cocktail becomes homogeneous in less than one minute. Time scale, 2 frame per second.
Case 3: An interferometric video recording an experiment consisting of pouring a lighter lysozyme protein solution (30 mg/ml) over a NaCl solution (3%). It is clearly observed the phenomenon of double-‐diffusive salt Ringering instability. The salt molecules diffuse faster into the pockets of proteins than the protein molecules into the pockets of salts. That simple mechanism creates density differences between the pockets and the surrounding solution that provokes the formation of upwards salt-‐rich Ringers and downwards protein-‐rich Ringering. Time scale, 2 frame per second.
Case 4: An interferometric video corresponding to an experiment consisting of pouring a Lysozyme protein solution (60 mg/ml) over a NaCl solution (9%). The interferometric mode switch to normal optical illumination between 27 and 34 seconds. Then, it can be clearly noticed that due to high concentration of the solutions, the Rluid is mixed by drag of the precipitated protein. Time scale, 2 frame per second.
t = 25.0 s t = 34.0 s t = 64.0 s t = 96.0 s t = 153.0 s
t = 0.0 s t = 3.0 s t = 10.0 s t = 16.0 s t = 20.0 s
Time sequence of interferograms. A solu,on of Lysozyme 30 mg/ml poured on top of a solu,on of NaCl 2% m/v.
Protein lighter more viscousNaCl heavier less viscous
As a pracBcal corollary of the results, it is recommended to perform mixing when seeking for reproducibility while avoid mixing when seeking for a more complex and extensive screening of the crystallisaBon space.
To Mix or not to Mix. Is that the ques7on?
From: On the mixing of protein crystallisa,on cocktailsEduardo I. Howard, José Miguel Fernandez and Juan Manuel Garcia-‐RuizSubmi]ed for publica,on
Batch Method
Batch Method no mixing = liquid-‐liquid diffusion
Capillary counter diffusion
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Requirements : Convection must be removed One reactant must flow faster than the other one It must be room to display the spatial pattern Initial conditions far from equilibrium
A precipitant chamber A long protein chamber
A physical buffer (optional)
The counter-‐diffusion technique
Based on diffusion–reaction patterns forming when two reactants are allowed to diffuse one against the other under initial conditions very far from equilibrium*
How do counter-‐diffusion works?
* J.M. Garcia-Ruiz, Counterdiffusion method for protein crystallisation. Methods in Enzimology 368 (2003) 130-154
tDdxerfcccppt
pptppt 221 0 +
= tDxerfcccp
pp 221 0 −
=The diffusion step: with Dppt >> Dp
The precipitation step is governed by supersaturation with respect to the solubility curve of the protein
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Counter-‐diffusion technique:How do it works?
Precipitant
Typical counterdiffusion crystallization patterns
Protein concentration
Precipitant concentration
Precipitant
Precipitant
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
A supersatura8on wave for protein crystallisa8on
Numerical simulaBon of the counter-‐diffusion technique
J.M. García-Ruiz, F. Otálora, M.L. Novella, J.A. Gavira, C. Sauter and O. Vidal. J. Crystal Growth 232 (2001) 149-155
F. Otálora and J.M. García-‐Ruiz. Computer simulaBon of the diffusion/reacBon interplay in the gel acupuncture method. Journal of Crystal Growth 169 (1996) 361
F. Otálora and J.M. García-‐Ruiz. Computer simulaBon of the diffusion/reacBon interplay in the gel acupuncture method. Journal of Crystal Growth 169 (1996) 361
Requirements : Convection must be removed One reactant must flow faster than the other one It must be room to display the spatial pattern Initial conditions far from equilibrium
A precipitant chamber A long protein chamber
A physical buffer (optional)
The counter-‐diffusion technique
Based on diffusion–reaction patterns forming when two reactants are allowed to diffuse one against the other under initial conditions very far from equilibrium*
tDdxerfcccppt
pptppt 221 0 +
= tDxerfcccp
pp 221 0 −
=The diffusion step: with Dppt >> Dp
The precipitation step is governed by supersaturation with respect to the solubility curve of the protein
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
NaCl
35 m
m
A demonstra8on of the dynamics of pa7ern forma8on in counterdiffusion technique
5 mm
[NaCl] 20% p/v[Agarose] 0.5% p/v[Lysozyme] 100 mg/ml[Agarose] 0.25% p/v
Advantage of CCD : Diffusive mixing assures reproducibility
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Precipitant concentration
Protein concentration
In batch + evaporation and vapor diffusion techniques the system moves towards far from equilibrium
In counter-diffusion technique the system moves from far from equilibrium to equilibrium:
The technique self-search the best crystallization conditions
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
How to perform counter-diffusion experiments?
23
forces drag viscousforcesbuoyancy −⋅⋅Δ⋅⋅== να gcLGrN
Microgravity981-9.8 x10-4 cm2 s-1
Gels (10-7-10-6 cm) and capillaries (2 x 10-2 cm)
Viscosity 0.015 g cm-1 s-1
Density gradient≈ 10-3 gr cm-3 mol-1
Looking for diffusion mass transport scenarios
J.M. García-‐Ruiz, J. Drenth, M. Ries-‐KauV and A. Tardieu. A world without gravity -‐ Research in space for health and industrial processes. Edited by G. Seibert et al., ESA SP 1251 June (2001)
At high GrN convecBon dominate
At low GrN convecBon dominate
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Volume of protein solution required to fill a capillary as a function of its
For capillaries of 0.1 mm internal diameter the required volume of protein solution is 314 nanoliters for 4 cm long capillaries and 235 nanoliters for 3 cm long capillaries. For a capillary diameter of 0,75 mm (the limit for a good visualization) the volume of protein solution is just 166 and 133 nanoliters respectively.
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capillary length = 3 cm capillary length = 4 cm capillary length = 5 cm
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Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Apoferri7n
A) Hfq, B) AP-‐Sm1, C) AF-‐Sm1 associated to a 14 bases RNA target RNA, D) EndoVII, E) EndoVII in complex with a DNA cruciform juncBon, F) EndoVII in complex with a mismatched DNA, G) lysozyme.
From: Sauter, Biertümpfel et al., Acta Cryst. (2002). D58, 1657
photosystem II core complex of Pisum sativumIvana Kuta et al., Photosynth Reserach 2007
Or by counterdiffusion techniques, since you hace already seen a good collec7on of crystal, here there are some more from other laboratory.
Structure of 21 simple mutants of tiorredoxine de E. Coli
native
Residuos cargados superficiales
Residuos hydrofobos enterrados
PDB I.D. 2FCH, 1ZZY, 2FD3, 2H6X, 2H6Y, 2H6Z, 2H70, 2H71, 2H72, 2H73, 2H74, 2H75 y 2H76
D EE DI VV I
Residue number20 40 60 80 100
ΔΔ
G (kJ/m
ol)
-5
-4
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A par7r de un análisis de secuencia se han determinado las mutaciones mas conservadas evolu7vamente. Para entender el por que de esta conservación se llevan a cabo estudios termodinámicos y estructurales que nos permitan en un futuro diseñar proteínas funcionales de mayor robustez.
SER38
Crystal structure of dihydropyrimidinase de S. meliloti...
…and mutants C76A y C181A of the hidantoin racemase of the
C76A C181A C76A-hidantoinaC76A-D,L-IPH C181A-D,L-MTEH
Dpto. Química, Área de Bioquímica y Biología Molecular. UAL. Dr. Felipe Rodríguez-Vico y Dr. Sergio Martínez-Rodríguez
N114A Abl-SH3 domain mutant complexed with a high affinity peptide ligand
Table 1.-‐ X-‐ray data collec7on sta7s7cs
Space group P212121
Unit cell dimensions 48.170 50.093 56.431 90.00 90.00 90.00
Resolution range (Å) 50-1.75
Number of observations 84702
Unique reflections 13236 (1638)
Data completeness (%) 92.2 (75.8)
Rmergeb (%) 0.07 (0.24)
I/σ(I) 16.4 (4.4)
a The values in parentheses are for the highest resolu7on bin
Camara-Artigas et al., Acta Cryst. (2007). D63, 646–652
A demonstration of the use of the method to solve structures at room temperature directly from 0.1 mm capillary screening.
In situ data collection and structure determination at room temperature
Protein Lysozyme Thaumatin HLFBPase FerritinMW 14,3 KDa 22 KDa 147 KDa 456 KDaIPtheoretical
9.3 8.5 6.6 5.4Precipitant NaCl Na/K Tart. PEG 4000 CdSO4pH 4.5 7.0 9.0 5.0
Protein and low concentration agarose
Precipitant + glycerol (cryo solution)
Crystal
Gavira, J.A. Et al.. (2002). Ab ini4o crystallographic structure determina,on of insulin form protein to electron density without crystal handling. Acta Crystallogr D58 :1085-‐1254Ng, J.D et al. (2003). Protein crystalliza,on by capillary counter-‐diffusion for applied crystallographic structure determina,on. Journal of Structural Biology 142:218-‐231.
Each protein was concurrently grown, deriva7zed with a halide, treated with a cryogenic solu7on in a single capillary tube and used directly for data collec7on using synchrotron radia7on source without ever handling the crystals.The structures were determined ab ini7o by Itera7ve Single Anomalous Sca[ering (ISAS) yielding to de novo crystallographic models. This procedure is proposed as a direct applica7on towards high thorough-‐put screening and structure determina7on for proteins in general.
Cryocrystallography with crystals grown by counterdiffusion techniqueKeeping crystals in the capillary (Method 1) In situ data collection and structure determination
XTALVIEW(Duncan E. McRee)
ISAS((Bi-Cheng Wang)
O(Alwyn Jones)
PHASES(W.Furey & S. Swaminathan)
XTALVIEW(Duncan E. McRee)
CCP4 (UK Science and Engineering Research Council)
+
J.A. Gavira, D. Toh, F.J. López-Jaramillo, J.M. García-Ruiz and J.D. Ng. Ab Initio crystallographic structure determination of insulin: from protein to electron density without crystal handling. Acta Cryst. D58 (2002) 1147-1154
SH3 Alpha-‐Spectrin pH 9.0mixPEGs
SH3 Alpha-‐Spectrin pH 5.0PEG8K
Example of capillary monted crystal for cryo-‐cooling
Spectrin-‐SH3Spectrin-‐SH3
RT 100K
Wavelength (Å) 0.977 0.977
Space group P212121 P212121
Cell parameters (Å) 34.46, 42.47, 50.04 33.66, 42.24, 49.73
Resolu7on limit (Å) 1.55 1.55
T data collec7on (K) RT 100
Precipitant mixPEGs mixPEGs
Crystalliza7on Technique CD Capillary CD Capillary
Crystalliza7on pH 9.0 9.0
Mosaicity 0.3-‐0.4 0.8-‐1.0
B factor 19.7 21.0
Example of a SH3 Alpha-‐Spectrin crystal collected at room temperature and then cryo-‐cooled at 100K.
SH3 Alpha-‐Spectrin pH 9.0mixPEGs
Precipitant/buffer cocktail
Gel plug
capillaries
cover
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Dip one capillary into the protein solution. The protein solution will rise by capillarity and the capillaries will be filled. Seal the upper end with the putty.
Dip the filled capillary into the GCB-Domino. Just punch the unsealed end of the capillary across the gel located on top of the precipitant.
J.D. Ng, R.C. Stevens and P.Kuhn, submiVed
Juan Ma. Garcia-Ruiz Tokyo 2nd International Symposium on Diffraction Structural Biology 2007
Low-cost plastic constructs fabricated from cyclic olefin polymers shown non-birrefringent and compatible materials for microfluidic crystallization.
Tested with bacteriorhodpsin with lysozyme, bacteriorodopsin and thaumatin
New technologies applied to counterdiffusion
In situ data collection and structure determination
Joseph D. Ng, Peter J. Clark, Raymond C. Stevens and Peter Kuhn. In situ X-‐ray analysis of protein crystals in low-‐birefringent and X-‐ray transmissive plasBc microchannels. Acta Cryst. (2008). D64, 189–197
Joseph D. Ng, Peter J. Clark, Raymond C. Stevens and Peter Kuhn. In situ X-‐ray analysis of protein crystals in low-‐birefringent and X-‐ray transmissive plasBc microchannels. Acta Cryst. (2008). D64, 189–197
Sauter et al., From macrofluidics to microfluidics for the crystallizaBon of biological macromolecules CGD 7 2007.
Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-‐772.
Using microfluidics to decouple nucleaBon and growth of protein crystals. J.U. Shim, G. Cristobal G, D.R. Link, T. Thorsen, S. Fraden, Crystal Growth & Design 7, 2192-‐2194 (2007).
Our aim is to reduce the screening for crystallizaBon condiBons as much as possible based on understanding
Could a mixture of PEGs do as well as each PEG separately?Is it the pH of the crystallizaBon experiment important when used salts as well as PEG as precipitaBng agent?
Study base on 20 proteins along one year:
PEG 400K (30%) ; six pH values PEG 4000 (PEG 30%); six pH values PEG 8000 (30 %); six pH values PEG 400K (20%) ; PEG 4000 (PEG 15%); PEG 8000 (10 %); six pH values
Ammonium sulphate 3M at six different pH values.
pH 4 pH 5 pH 6 pH 7 pH 8 pH 9
The precipitant cocktail contains:
a) A buffer that keep the solution within one unit of pHb) A low molecular weight polyethylene glycol c) A high molecular weight polyethylene glycold) An additive that may help to precipitate the protein.
)(TRTD B
ηπκ
≅According to Einstein-Stokes relation the diffusivity depends on the radius of the molecule. Therefore:
Buffer molecules diffuses faster and change the pH of the protein solutionLow MW PEG molecules go later onFinally high MW PEG molecules scan the capillary
Several effects in one single experiment !All concentrations tested in one experiment !
Juan Ma. Garcia-‐Ruiz XII InternaBonal Conference on the CrystallizaBon of Biological Macromolecules Cancun, 6-‐9 of May, 2008
Crystallizability is very sensiBve to pH –as expected. The bad new is that there is not A clear correlaBon with pI. When crystals form at any pH, precipitaBon occurs in any other pH
Crystallizability is sensiBve to pH. There is not a clear correlaBon with pI.The 3PEG mixture does as well as each of them. However , it is required to increase the concentraBon of PEG 8K
Some additional results observed :
1. It is confirmed that, for the same screening matrix, counterdiffusion yields at least as many hits as vapor diffusion, within the first three weeks after set-up of the experiments.
2. It is confirmed that for similar crystallization conditions, nucleation is retarded in the case of counterdiffusion with respect to drop techniques.
3. It has been also proven that counterdiffusion yields crystals under screening conditions that drop techniques never produced. Noticeably the number of new crystallization conditions increases with time, sometime with waiting periods of month(S)
crystal form changes with crystallisation technique The way in which supersaturation is achieved has an effect on the crystal form. Protein crystallisation crystal form technique
Cablys3*lysozyme hanging drop PEG P212121 67 69 113 90 90 90 hanging drop formate C2 133 73 39 90 105 90 APCF C2 129 73 38 90 107 90 counterdiffusion formate P212121 68 69 113 90 90 90
TIM hanging drop AS P3221 125 104 counterdiffusion AS P3x12 215 106
Parvalbumin sitting drop P212121 51 50 35 counterdiffusion P21212 59 59 26 counterdiffusion P1 26 32 53 85 83, 79
From I. Zeggers, J.M. García-Ruiz and coworkers, unpublished data
PDB ID Variant S G Condition Method1KEB P40S. P 1 pH: 3.8
Ethanol: 25% CuAc2:10 mMNaAc:100 mM
Diffusion-Vd
1SRX Forma oxidada C 2 pH: 3.8Ethanol: 25%CuAc2:10 mMNaAc: 100 mM
Diffusion-Vd
2TRX Silvestre C 2 T: 4° CpH: 3.7-4.1Butane 0.001-0MMPD: 48-50%NaAc: 0.01-0MCuAc2:1 mM
Micro-dialysis
2FCH G74S P 21 21 21 T: 4° CpH: 5.4 MPD: 60%NaAc: 15 mMCuAc2: 1 mM
Counter-diffusion
1ZZY L7V P1 pH: 3.8Ethanol: 25%CuAc2: 10 mMNaAc: 100 mM
Counter-diffusion
2FD3 P34H P1 21 1 T: 4° CpH: 8.0 MPD: 60%Hepes: 15 mMCuAc2:1 mM
Counter-diffusion
2H6X2H6Y a Z2H70 a 76
WildE48D, E44DD9E,D47E, E85DD43E, D2E, D13ED10E
P61 pH: 6.9pH:3.5, 7.0 pH: 7.0, 7.0, 5.4pH:7.0, 3.5, 7.0pH:4.1T: 4° CMPD: 60% NaAc/Hepes:15mMCuAc2:1 mM
Counter-diffusion
Counterdiffusion produces new polymorphs
Vrije Universiteit Brussel, Université de Liege, Univesrité Catolique de Louvaine
Screening with counter-diffusion technique
Simple and fast preparation of the experiments
No tools required. Just fill the capillary and punch it into the precipitation cocktail
Extensive search of the crystallization space for pH and precipitant concentrations
Actual grid screen can be made possible and are recomended
Requires minimal amount of protein
Even lest than 250 nanoliters per capillary.
No pictures and image analysis required.
The whole growth history is stored in the capillaries.
Unambiguous interpretation of the results
The technique does not yield single results but a trend
The number of conditions for effective screening can be reduced
Mix of PEGs can be used to replace singular MW PEGS.
Coauthors in the work presented in this talk:
Luis Antonio Gonzalez: CrystallizaBon experiment implementaBonJosé Luis Gavira: Handling of crystals and X-‐ray diffracBon analysisEduardo Howards: Work on mixing experimentsJoe Ng: First screening and in situ X-‐ray diffracBon analysisFermín Otalora: Computer simulaBons
Financial support from and help from:
European Community VI Framework ProgramSpanish Government (MEC)Triana Science and TechnologyThis is a product of La Factoría de Cristalización
Thank you
This is a product of La Factoría de Cristalización