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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