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Non standard techniques for tackling the bottleneck of protein crystallization

Non standard techniques for tackling the bottleneck of protein crystallization. Stockholm, Sweden April 25,2007. Standard methods. Vapour diffusion-by far the most widely employed crystallisation method. Batch crystallisation-the oldest method.

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Non standard techniques for tackling the bottleneck of protein crystallization

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

  2. 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)

  3. Crystallisation Phase Diagram A. Batch B. Vapour diffusion C. Dialysis D. Free interface diffusion

  4. 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.

  5. Contribution of Oil to Macromolecular Crystallisation • Accuracy and reproducibility • Cleanliness • Control of Nucleation • Protection of Sample • Stability of Crystals

  6. Application of oils: • Screening • Optimisation • Control of nucleation and equilibration

  7. Oils for Optimisation Actively control the crystallisation environment as the trial takes place Lead crystal growth in desired direction

  8. 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 (Al’s oil) Nature of oil D'Arcy et al Journal of Crystal Growth,168, 175-180.

  9. 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.

  10. 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

  11. Schematic representation of dispensing drop under oil

  12. Trays used in microbatch crystallisation

  13. IMPAX Robot

  14. Protein crystals grown under oil

  15. …and more

  16. β-crustacyanin crystal Cianci et al (2002) PNAS 99, 9795-9800

  17. 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)

  18. Crystallisation of Membrane Proteins in oil using a variety of detergents • Dodecyl-b-d-maltoside • n-decyl-b-d-maltoside • n-decyl-b-d-glucopyranoside • Triton X-100 • n-nonyl-b-d-glucopyranoside • Sulphobetaine-14, sulphobetaine-12 • n-octyl-b-d-glucopyranoside • N,N-dimethyl-dodecylamine oxide Nield, J.M. (1997) PhD thesis Imperial College

  19. Yeast F1c10 crystals From Stock, Leslie and Walker Stock et al

  20. CP 43 Crystals Screening Fine-tuning .

  21. Problems Associated with Microbatch Crystallisation • Shock nucleation • Use of small volatile organic components

  22. NUCLEATION • 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...

  23. Factors influencing Nucleation • Concentration of precipitant, protein, additives • Temperature, pH • Biological contaminants • Minerals • Dust • Container walls • Speed of equilibration- NA to batch • Seeds • Microcrystals • Macrocrystals

  24. Create environment to limit number of nuclei ‘Back off’ from nucleation conditions to metastable Bypass the nucleation zone Strategies for obtaining few high quality crystals Chayen, N. E. (2004) Curr Opin Struct Biol,14, 577-83.

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

  26. Containerless vs. Contact

  27. CG2 Crystals

  28. Original containerless not HTP! • Fiddly to layer the oils • Drop size of ≥ 2 µl • Migration of drops to walls • Difficult to harvest crystals • Not amenable to automation

  29. Insertion of Oil Barrier in Hanging Drops Chayen, N. E. (2005). Prog Biophys Mol Biol,88, 329-37.

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

  31. Improvement due to oil barrier

  32. Improvement due to oil barrier Mandelman, D.et al (2002) Acta Crystallogr D Biol Crystallogr,58, 668-71.

  33. Limitations of oil barrier method • Does not work if reservoir contains PEG or MPD above 15%

  34. (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 (c) Crystals grown by transfer from nucleation to growth conditions Improvement of crystal quality by separation of Nucleation and Growth Saridakis, E. and Chayen, N. E. (2003) Biophys J,84, 1218-22.

  35. Practical Separation of Nucleation and Growth Saridakis and Chayen (2000) Prot. Sci. 9, 755-757

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

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