Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart

1. Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart Imperial College, London: Professor David M. Blow, Patrick Shaw Stewart, Dennis Maeder, Naomi Chayen Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, James Smith, Richard Briggs

2. What is microbatch crystallization? • Crystallization in small drops under oil

3. (2-bore) microtip Oil Sample Microbatch crystallization

4. Microbatch crystallization

5. Microbatch crystallization

6. End of a 5-bore microtip 0.15 mm 0.9 mm

7. Microbatch optimization – print out

8. ORYX 6 crystallization system

9. Oryx Crystallization System • Versatile – optimization, additives etc.

10. Oryx Crystallization System • Versatile – optimization, additives etc. • Wastes very little protein (around 0.5 µl per experiment)

11. Oryx Crystallization System • Sitting drops – 0.1+0.1 to 1+1µ

12. Oryx Crystallization System • Sitting drops – 0.1+0.1 to 1+1µ • Microbatch screening – 0.1+0.1 to 1+1µl

13. Oryx Crystallization System • Sitting drops – 0.1+0.1 to 1+1µ • Microbatch screening – 0.1+0.1 to 1+1µl • Microbatch optimization – 0.5+0.5to1+1µl

14. How does Oryx work? 2-bore Microtip – screening 5-bore Microtip – optimization

15. Sitting Drop

16. Sitting Drop - preparation • Use a 2-bore microtip

17. Sitting Drop - preparation • Use a 2-bore microtip • Start with both bores full of water

18. Sitting Drop - preparation • Use a 2-bore microtip • Start with both bores full of water • Suck up 1µl of air into both channels Air bubbles

19. Sitting Drop - preparation Air bubble • Use a 2-bore microtip • Start with both bores full of water • Suck up 1µl of air into both channels • Suck up protein required for experiment + 0.25 µl Protein slug

20. Sitting Drop – dispensing cycle • Rinse in reservoir (1)

21. Sitting Drop – dispensing cycle • Rinse in reservoir • Move sideways and pick up clean solution (1) (2)

22. Sitting Drop – dispensing cycle • Rinse in reservoir • Move sideways and pick up clean solution • Dispense solution and protein (3) (1) (2)

23. Hardware

24. IMPAX 1-5 crystallization system

25. Liquid-handling channel • Motorized Hamilton gas-tight syringe (water) • Ground-glass syringe (water) • Valve • Disposable syringe (reagents) • Valve • Microtip X 5

26. IMPAX 1-5 crystallization system

27. ORYX 6 crystallization system

28. ORYX 6 crystallization system

29. Microbatch screening

30. Microbatch screening – dispensing cycle Target plate Screening solutions

31. Microbatch screening – dispensing cycle • Pick up screening solution (1)

32. Microbatch screening – dispensing cycle • Pick up screening solution • Transfer to microbatch drop (1) (2)

33. Microbatch screening – dispensing cycle • Pick up screening solution • Transfer to microbatch drop; oil (1) (2) + oil

34. Microbatch screening – dispensing cycle • Pick up screening solution • Transfer to microbatch drop; oil • Rinse (1) (2) (3) + oil

35. Microbatch optimization

36. Microbatch optimization – dispensing cycle • Dispense five solutions together (1)

37. Microbatch optimization – dispensing cycle • Dispense five solutions together • Oil (1) + oil

38. Microbatch optimization – dispensing cycle • Dispense five solutions together • Oil (1) + oil

39. Microbatch optimization – dispensing cycle • Dispense five solutions together • Oil (1) + oil

40. Phase diagram of a protein precipitate [Protein] clear [Precipitant]

41. Phase diagram of a protein precipitate nucleation [Protein] clear [Precipitant]

42. precipitate nucleation [Protein] metastable zone clear [Precipitant] Phase diagram of a protein

43. Phase diagram of a protein p n [Protein] m.z. Vapor diffusion c [Precipitant]

44. Phase diagram of a protein p n Microbatch [Protein] m.z. v.d. c [Precipitant]

45. Phase diagram of a protein p n M.B.(paraffin) [Protein] m.z. v.d.. M.B.(par./si.) c [Precipitant]

46. Phase diagram of a protein p n M.B.(paraffin) OPTIMIZATION [Protein] m.z. v.d. M.B.(par./si.) SCREENING [Precipitant]

47. What % of protein should you use? Microbatch with Si. / Par.: n [Protein] Protein stock m.z. 50% Precipitant saturated Precipitant stock [Precipitant]

48. What % of protein should you use? Microbatch with Si. / Par.: n [Protein] Protein stock m.z. 66% 50% Precipitant saturated Precipitant stock [Precipitant]

49. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm

50. Screening: studies comparing microbatch with vapor diffusion P.F.M. Baldock, V. Mills, P.D. Shaw Stewart. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. Journal of Crystal Growth. 168 (1996), pp 170-174 or: http://www.douglas.co.uk/rep2.htm A. D’Arcy, G.E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000.