Non standard techniques for tackling the bottleneck of protein crystallization Stockholm, Sweden Stockholm, Sweden April 25,2007

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  • Non standard techniques for tackling the bottleneck of protein crystallization Stockholm, Sweden Stockholm, Sweden April 25,2007
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  • Standard methods Vapour diffusion-by far the most widely employed crystallisation method. Batch crystallisation-the oldest method. Other diffusion methods like Dialysis, Free Interface Diffusion (FID)
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  • Crystallisation Phase Diagram A. Batch B. Vapour diffusion C. Dialysis D. Free interface diffusion
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  • Problems with Vapour Diffusion Changes in drop volume. Changes in pH due to volatile ions. Condensation with slight temperature change dissolution of crystals. Composition changing during crystallisation process.
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  • Contribution of Oil to Macromolecular Crystallisation Accuracy and reproducibility Cleanliness Control of Nucleation Protection of Sample Stability of Crystals
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  • Application of oils: Screening Optimisation Control of nucleation and equilibration
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  • Oils for Optimisation Actively control the crystallisation environment as the trial takes place Lead crystal growth in desired direction
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  • Nature of oil Different oils can be used to control the rate of evaporation Silicone fluid-a polymeric compound of dimethyl siloxane units Paraffin oil-mineral oil of branched paraffins in the C20+ range. A mixture of silicone and paraffin oil (Als oil) D'Arcy et al Journal of Crystal Growth, 168, 175-180.
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  • Advantages of Crystallisation under Oil Some crystals will only grow in oil. Hanging drops tend to spread over the surface of siliconized cover slips because of the decrease in surface tension caused by the detergent. In batch drops are round an symmetrical. Mechanically batch is simplest crystallization Lends itself readily to HTP. Chayen, N. E. (1997) Structure, 5, 1269-74. Chayen, N. E. (1997) Structure, 5, 1269-74.
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  • MICRO-BATCH CRYSTALLISATION Miniaturized version of Batch. The technique makes use of low density oil as the medium. Supersaturation is reached upon mixing thus there is no change in the conditions once the experiment has been dispensed. Chayen et al (1990) Journal of Applied Crystallography, 23, 297-302
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  • Schematic representation of dispensing drop under oil
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  • Trays used in microbatch crystallisation Trays used in microbatch crystallisation
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  • IMPAX Robot
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  • Protein crystals grown under oil
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  • and more
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  • -crustacyanin crystal Cianci et al (2002) PNAS 99, 9795-9800
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  • Examples of Membrane Proteins crystallised in Microbatch under oil Outer membr phosphorilase A (Schneider) Photosystem I ( Nechushtai ) CP43 ( Henkamer) LHC II from Rhodopseudomonas acidophila ( McDermot ) F1C10 of ATP Synthase (Stock, Leslie, Walker) P-glycoprotein (Rosenberg ) MRP1 ( Rosenberg )
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  • Crystallisation of Membrane Proteins in oil using a variety of detergents Dodecyl--d-maltoside n-decyl--d-maltoside n-decyl--d-glucopyranoside Triton X-100 n-nonyl--d-glucopyranoside Sulphobetaine-14, sulphobetaine-12 n-octyl--d-glucopyranoside N,N-dimethyl-dodecylamine oxide Nield, J.M. (1997) PhD thesis Imperial College
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  • Yeast F1c10 crystals Stock et al From Stock, Leslie and Walker
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  • CP 43 Crystals Screening Fine-tuning.
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  • Problems Associated with Microbatch Crystallisation Shock nucleation Use of small volatile organic components
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  • Key to crystallisation and is poorly controlled. Pre-requisite and first step that determines the crystallization process and its result. Ability to control it would give us a handle on the crystallization process... NUCLEATION
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  • Factors influencing Nucleation Concentration of precipitant, protein, additives Concentration of precipitant, protein, additives Temperature, pH Temperature, pH Biological contaminants Biological contaminants Minerals Minerals Dust Dust Container walls Container walls Speed of equilibration- NA to batch Speed of equilibration- NA to batch Seeds Seeds Microcrystals Microcrystals Macrocrystals Macrocrystals
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  • Strategies for obtaining few high quality crystals Create environment to limit number of nuclei Back off from nucleation conditions to metastable Bypass the nucleation zone Chayen, N. E. (2004) Curr Opin Struct Biol, 14, 577-83.
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  • Containerless crystallisation Chayen, N. E. (1996) Protein Engineering, 9, 927-929
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  • Containerless vs. Contact
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  • CG2 Crystals
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  • Original containerless not HTP! Fiddly to layer the oils Drop size of 2 l Migration of drops to walls Difficult to harvest crystals Not amenable to automation
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  • Insertion of Oil Barrier in Hanging Drops Chayen, N. E. (2005). Prog Biophys Mol Biol, 88, 329-37.
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  • Optimisation of lobster protein crystals using an oil barrier in hanging drops
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  • Improvement due to oil barrier
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  • Mandelman, D.et al (2002) Acta Crystallogr D Biol Crystallogr, 58, 668-71.
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  • Limitations of oil barrier method Does not work if reservoir contains PEG or MPD above 15%
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  • Improvement of crystal quality by separation of Nucleation and Growth (a) Working phase diagram of c-phycocyanin. Arrows correspond to transfer from spontaneous nucleation conditions (black circles) to optimal growth conditions (open circles). (a) Working phase diagram of c-phycocyanin. Arrows correspond to transfer from spontaneous nucleation conditions (black circles) to optimal growth conditions (open circles). (b) Crystals grown by standard vapour diffusion trials (b) Crystals grown by standard vapour diffusion trials (c) Crystals grown by transfer from nucleation to growth conditions (c) Crystals grown by transfer from nucleation to growth conditions Saridakis, E. and Chayen, N. E. (2003) Biophys J, 84, 1218-22.
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  • Practical Separation of Nucleation and Growth Saridakis and Chayen (2000) Prot. Sci. 9, 755-757
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  • Transfer From Krengel, U. et al. Acta Cryst. (2006) F62, 441-445