crystallization methods and...

Juan Manuel García-Ruiz

Laboratorio de Estudios CristalográficosCSIC-Universidad de Granada, Spain

[email protected]

Crystallization Methods and Screening

viernes 14 de octubre de 2011

mailto:[email protected]

mailto:[email protected]

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

Juan Ma. Garcia-‐Ruiz XII Interna3onal Conference on the Crystalliza3on of Biological Macromolecules Cancun, 6-‐9 of May, 2008


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)



The batch method

Mixing solvents and/or precipitating agents to obtain solutions in the metastable region


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


Technical details:

Drops are usually of the size of microliters Drops as small as few nanoliters can be used Remember that the number of nucleation events depends on the drop volume Non-crystallizing drops can be allowed to evaporate To slow down evaporation, drops can be covered by oil or immersed into oils Be sure to mix well protein and precipitating agent There are different devices available to perform batch experiments, see for instance the web page of , Hampton Research, Macromolecular solutions and Jena Biosciences In principle, you can use any small reservoir than can be sealed and can be observed with a binocular microscope.


The batch method and evaporation

Mixing solvents and/or precipitating agents to obtain solutions in the undersaurated region and move it to the metastable region

Recommendations: Never use large volume of solution. Try to use drops smaller than 5 microlitersThis will save sample, will reduce convection, will reduce surface tension induced problems and will avoid production of microcrystals in the air/liquid interface.Use oils to control evaporationAvoid evaporation in closed room or closed space (refrigerators, incubators) and in rooms without a good air exchange system


The vapour diffusion method

Hanging drop (and sitting drop) technique

Master de Cristalogra.ía y Cristalización CSIC-UIMP Juan Ma. Garcia-Ruiz


Vapour diffusion exchange of solvents

V2

The antisolvent must be more volatile that the solvent

V1 > V2

V1 V1 > V2

Solution of the

compound in solvent 2Antisolvent


0 5 10 15 20 25 30 35 40 45 500

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E'

0'

Understurated zone

Metastable zone

Labile zone

0

E

b) Vapour diffusion experiment

Mac

rom

olec

ule

conc

entra

tion

Precipitating agent concentration

Vapour diffusionHanging/SiKng drop

Protein solution + precipitant at low concentration

Precipitant at high concentration(usually twice the one in the drop)



0 5 10 15 20 25 30 35 40 45 500

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Metastable zone

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Mac

rom

olec

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tion


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 …


Transport of water molecules

Crystallisationin the drop




0 5 10 15 20 25 30 35 40 45 500

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Labile zone

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Mac

rom

olec

ule

conc

entra

tion


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 …


Transport of water molecules

Crystallisationin the drop



Note that the solu3on moves from equilibrium to out of equilibrium:It means ini3al condi3ons and/or rate of evapora3on need to be tuned


0 1 2 3 4 5 6 7 8 9 100

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Pro

tein

con

cent

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n (m

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l)

NaCL (%w/v)


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50

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tein

con

cent

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Changing the initial concentrations (in the well and in the drop) the rate of supersaturation can be selected


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Pro

tein

con

cent

ratio

n (m

g/m

l)

NaCL (%w/v)

Number of visited crystallization conditions per trial

• Different supersaturation values from equilibrium to out of equilibrium

• One (variable) rate of supersaturation

Changing the initial concentrations (in the well and in the drop) the rate of supersaturation can be selected


The final concentration of precipitant agent and of protein in the drop can be easily calculated.

2

1

2XX

Final concentrationof precipitating agentin the drop

Starting concentrationof precipitating agent in the drop

Final concentrationof protein in the drop

Staring concentrationof protein in the drop

Prot

ein

Precipitating agent



Technical details:

Drops are usually of the size of 2- 20 microliters Drops as small as few hundred nanoliters can be used Remember that the number of nucleation events depends on the drop volume Non-crystallizing drops can be allowed to evaporate Siliconised cover slides avoid hetreogeneous nucleation and sticking of crystals to the glass Be sure to seal properly the crystallization system to avoid evaporation by equilibration with the atmosphere. There are different devices available to perform vapor diffusion experiments, see for instance the web page of , Hampton Research, Macromolecular solutions and Jena Biosciences. Most of them are based on 24 or 96 wells configuration of Limbro-like plates, but other devices are also available.There are no differences between sitting and hanging drop methods from the point of view of crystallization kinetics. The parameter controlling the evaporation of the drop is the distance drop-precipitant surface.


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


A crystallizing drop is not an homogeneous systemSmaller the drop smaller the number of nuclei. Large drop may give crystals of different quality

Reminding Mark



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 \irst 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 solu3on = density precipitant solu3on

Protein heavier

Protein lighter

Rela<ve viscosi<es 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 \ingering 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 \ingers and downwards protein-‐rich \ingering. 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 \luid 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 prac<cal 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 crystallisa<on space.

To Mix or not to Mix. Is that the ques3on?

From: On the mixing of protein crystallisa6on cocktailsEduardo I. Howard, José Miguel Fernandez and Juan Manuel Garcia-‐RuizCrystal Growth & Design (2010)


Batch Method

Batch Method no mixing = liquid-‐liquid diffusion

Capillary counter diffusion



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 +

=tD

xerfcccp

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



Counter-‐diffusion technique:How do it works?

Precipitant

Protein concentration

Precipitant concentration




Precipitant

Typical counterdiffusion crystallization patterns






Precipitant

Typical counterdiffusion crystallization patterns



Precipitant

Precipitant



A supersatura8on wave for protein crystallisa8on

Numerical simulaAon 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 simula3on of the diffusion/reac3on interplay in the gel acupuncture method. Journal of Crystal Growth 169 (1996) 361

F. Otálora and J.M. García-‐Ruiz. Computer simula3on of the diffusion/reac3on 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 +

=tD

xerfcccp

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



NaCl

35 m

mA 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





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



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-‐Kau_ 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 convec3on dominate

At low GrN convec3on dominate



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 diameter (microns)

capillary length = 3 cm capillary length = 4 cm capillary length = 5 cm

20 40 60 80 1000

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Apoferri)n


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 junc3on, 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


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

-3

-2

-1

0

1

2

85

60

41

38

23

55

45

91

7572

13

2

4347

9

10

4

5 16

25

3061

44

48

101

10486


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


N114A Abl-SH3 domain mutant complexed with a high affinity peptide ligand

Table 1.-‐ X-‐ray data collec<on sta<s<cs

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 resolu<on 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 ini&o 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, deriva<zed with a halide, treated with a cryogenic solu<on in a single capillary tube and used directly for data collec<on using synchrotron radia<on source without ever handling the crystals.The structures were determined ab ini<o by Itera<ve Single Anomalous ScaZering (ISAS) yielding to de novo crystallographic models. This procedure is proposed as a direct applica<on towards high thorough-‐put screening and structure determina<on 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

Resolu)on limit (Å) 1.55 1.55

T data collec)on (K) RT 100

Precipitant mixPEGs mixPEGs

Crystalliza)on Technique CD Capillary CD Capillary

Crystalliza)on 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



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, submi_ed

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 plas3c 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 plas3c microchannels. Acta Cryst. (2008). D64, 189–197


Sauter et al., From macrofluidics to microfluidics for the crystalliza3on 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 nuclea3on 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 crystalliza<on condi<ons 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 crystalliza<on experiment important when used salts as well as PEG as precipita<ng 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:



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 !



Crystallizability is very sensi<ve to pH –as expected. The bad new is that there is not A clear correla<on with pI. When crystals form at any pH, precipita<on occurs in any other pH


Crystallizability is sensi3ve to pH. There is not a clear correla3on with pI.The 3PEG mixture does as well as each of them. However , it is required to increase the concentra3on 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.


crystallization methods and...

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