Non standard techniques Non standard techniques for tackling the bottleneck for tackling the bottleneck of protein crystallizationof protein crystallization

Stockholm, SwedenStockholm, SwedenApril 25,2007April 25,2007

Standard methodsStandard methods

Vapour diffusion-by far the most Vapour diffusion-by far the most widely employed crystallisation widely employed crystallisation method.method.

Batch crystallisation-the oldest Batch crystallisation-the oldest method.method.

Other diffusion methods like Dialysis, Other diffusion methods like Dialysis, Free Interface Diffusion (FID)Free Interface Diffusion (FID)

Crystallisation Phase DiagramCrystallisation Phase Diagram

A. BatchB. Vapour diffusion

C. DialysisD. Free interface diffusion

Problems with Vapour Problems with Vapour DiffusionDiffusion

Changes in drop volume.Changes in drop volume.

Changes in pH due to volatile ions.Changes in pH due to volatile ions.

Condensation with slight temperature Condensation with slight temperature

change change dissolution of crystals.dissolution of crystals.

Composition changing during Composition changing during

crystallisation process.crystallisation process.

Contribution of Oil to Contribution of Oil to Macromolecular CrystallisationMacromolecular Crystallisation

Accuracy and Accuracy and

reproducibilityreproducibility

CleanlinessCleanliness

Control of NucleationControl of Nucleation

Protection of SampleProtection of Sample

Stability of CrystalsStability of Crystals

Application of oils:Application of oils:

ScreeningScreening

OptimisationOptimisation

Control of nucleation and equilibrationControl of nucleation and equilibration

Oils for OptimisationOils for Optimisation

Actively control the crystallisation Actively control the crystallisation environment as the trial takes placeenvironment as the trial takes place

Lead crystal growth in desired directionLead crystal growth in desired direction

Nature of oilNature of oil

Different oils can be used Different oils can be used to control the rate of to control the rate of evaporationevaporation

Silicone fluid-a polymeric Silicone fluid-a polymeric compound of dimethyl compound of dimethyl siloxane unitssiloxane units

Paraffin oil-mineral oil of Paraffin oil-mineral oil of branched paraffins in the branched paraffins in the C20+ range.C20+ range.

A mixture of silicone and A mixture of silicone and paraffin oil (Al’s oil)paraffin oil (Al’s oil)

D'Arcy et al D'Arcy et al Journal of Crystal Growth,Journal of Crystal Growth, 168,168, 175-180. 175-180.

Advantages of Crystallisation Advantages of Crystallisation under Oilunder Oil

Some crystals will only grow in oil.Some crystals will only grow in oil.

Hanging drops tend to spread over the surface of Hanging drops tend to spread over the surface of siliconized cover slips because of the decrease in siliconized cover slips because of the decrease in surface tension caused by the detergent. In batch surface tension caused by the detergent. In batch drops are round an symmetrical.drops are round an symmetrical.

Mechanically batch is simplest crystallization Mechanically batch is simplest crystallization Lends itself readily to HTP.Lends itself readily to HTP.

Chayen, N. E. (1997) Chayen, N. E. (1997) Structure,Structure, 5,5, 1269-74 1269-74..

MICRO-BATCH CRYSTALLISATIONMICRO-BATCH CRYSTALLISATION

Miniaturized version of Batch. Miniaturized version of Batch. The technique makes use of low The technique makes use of low

density oil as the medium.density oil as the medium. Supersaturation is reached upon Supersaturation is reached upon

mixing thus there is no change in the mixing thus there is no change in the conditions once the experiment has conditions once the experiment has been dispensed.been dispensed.

Chayen et al (1990) Journal of Applied Crystallography, 23, 297-302

Schematic representation of Schematic representation of dispensing drop under oildispensing drop under oil

Trays used in microbatch Trays used in microbatch crystallisationcrystallisation

IMPAX RobotIMPAX Robot

Protein crystals grown under oilProtein crystals grown under oil

……and moreand more

ββ-crustacyanin crystal-crustacyanin crystal

Cianci et al (2002) PNAS 99, 9795-9800

Examples of Membrane Proteins Examples of Membrane Proteins crystallised in Microbatch under crystallised in Microbatch under

oiloil Outer membr phosphorilase A Outer membr phosphorilase A (Schneider)(Schneider)

Photosystem I Photosystem I ((NechushtaiNechushtai)) CP43 (CP43 (Henkamer)Henkamer)

LHC II from Rhodopseudomonas acidophila LHC II from Rhodopseudomonas acidophila ((McDermotMcDermot))

F1C10 of ATP Synthase F1C10 of ATP Synthase (Stock, Leslie, Walker)(Stock, Leslie, Walker)

P-glycoprotein P-glycoprotein (Rosenberg(Rosenberg)) MRP1 (MRP1 (RosenbergRosenberg))

Crystallisation of Membrane Proteins Crystallisation of Membrane Proteins in oil using a variety of detergentsin oil using a variety of detergents

Dodecyl-Dodecyl--d-maltoside-d-maltoside n-decyl-n-decyl--d-maltoside-d-maltoside n-decyl-n-decyl--d-glucopyranoside-d-glucopyranoside Triton X-100Triton X-100 n-nonyl-n-nonyl--d-glucopyranoside-d-glucopyranoside Sulphobetaine-14, sulphobetaine-12Sulphobetaine-14, sulphobetaine-12 n-octyl-n-octyl--d-glucopyranoside-d-glucopyranoside N,N-dimethyl-dodecylamine oxideN,N-dimethyl-dodecylamine oxide

Nield, J.M. (1997) PhD thesis Imperial College

Yeast F1c10 crystalsYeast F1c10 crystals

From Stock, Leslie and Walker

CP 43 CrystalsCP 43 Crystals

Screening Fine-tuning

.

Problems Associated with Problems Associated with Microbatch CrystallisationMicrobatch Crystallisation

Shock nucleation Use of small volatile organic

components

Key to crystallisation and is poorly Key to crystallisation and is poorly controlled.controlled.

   Pre-requisite and first step that Pre-requisite and first step that

determines the crystallization process determines the crystallization process and its result.and its result.

Ability to control it would give us a Ability to control it would give us a handle on the crystallization process...handle on the crystallization process...

NUCLEATIONNUCLEATION

Factors influencing NucleationFactors influencing Nucleation

Concentration of precipitant, protein, additivesConcentration of precipitant, protein, additives Temperature, pHTemperature, pH Biological contaminants Biological contaminants MineralsMinerals DustDust Container wallsContainer walls Speed of equilibration- NA to batchSpeed of equilibration- NA to batch Seeds Seeds

• MicrocrystalsMicrocrystals• MacrocrystalsMacrocrystals

Strategies for obtaining few Strategies for obtaining few high quality crystalshigh quality crystals

Create environment Create environment

to limit number of to limit number of

nucleinuclei

‘‘Back off’ from Back off’ from

nucleation conditions nucleation conditions

to metastableto metastable

Bypass the nucleation Bypass the nucleation

zonezone

Chayen, N. E. (2004) Curr Opin Struct Biol, 14, 577-83.

Containerless crystallisationContainerless crystallisation

Chayen, N. E. (1996) Protein Engineering, 9, 927-929

Containerless vs. ContactContainerless vs. Contact

CG2 CrystalsCG2 Crystals

Original containerless not HTP!Original containerless not HTP!

Fiddly to layer the oilsFiddly to layer the oils Drop size of ≥ 2 Drop size of ≥ 2 µlµl Migration of drops to wallsMigration of drops to walls Difficult to harvest crystalsDifficult to harvest crystals Not amenable to automationNot amenable to automation

Insertion of Oil Barrier in Hanging DropsInsertion of Oil Barrier in Hanging Drops

Chayen, N. E. (2005). Prog Biophys Mol Biol, 88, 329-37.

Optimisation of lobster protein crystals Optimisation of lobster protein crystals using an oil barrier in hanging dropsusing an oil barrier in hanging drops

Improvement due to oil barrierImprovement due to oil barrier

Improvement due to oil barrierImprovement due to oil barrier

Mandelman, D.et al (2002) Acta Crystallogr D Biol Crystallogr, 58, 668-71.

Limitations of oil barrier methodLimitations of oil barrier method

►Does not work if reservoir Does not work if reservoir contains PEG or MPD above 15%contains PEG or MPD above 15%

Improvement of crystal quality by separation Improvement of crystal quality by separation of Nucleation and Growthof Nucleation and Growth

(a) Working phase (a) Working phase diagram of c-diagram of c-phycocyanin. Arrows phycocyanin. Arrows correspond to transfer correspond to transfer from spontaneous from spontaneous nucleation conditions nucleation conditions (black circles) to optimal (black circles) to optimal growth conditions (open growth conditions (open circles). circles).

(b) Crystals grown by (b) Crystals grown by standard vapour standard vapour diffusion trialsdiffusion trials

(c) Crystals grown by (c) Crystals grown by transfer from nucleation transfer from nucleation to growth conditionsto growth conditions

Saridakis, E. and Chayen, N. E. (2003) Saridakis, E. and Chayen, N. E. (2003) Biophys J,Biophys J, 84,84, 1218-22 1218-22..

Practical Separation of Nucleation Practical Separation of Nucleation and Growthand Growth

Saridakis and Chayen (2000) Prot. Sci. 9, 755-757

Transfer

From Krengel, U. et al. Acta Cryst. (2006) F62, 441-445