1. Douglas Instruments Microbatch seminar- slide 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, Vaughan Mills, James Smith

2. Douglas Instruments Microbatch seminar- slide 2 1.What is the microbatch method? 2.Hardware and dispensing routines 3.Phase diagrams 4.Comparisons of microbatch and vapor diffusion 5.Case studies 6.Harvesting crystals 7.Experimental design

3. Douglas Instruments Microbatch seminar- slide 3 1.What is the microbatch method? 2.Hardware and dispensing routines 3.Phase diagrams 4.Comparisons of microbatch and vapor diffusion 5.Case studies 6.Harvesting crystals 7.Experimental design

4. Douglas Instruments Microbatch seminar- slide 4 What is the microbatch method? Crystallization in small drops under oil

5. Douglas Instruments Microbatch seminar- slide 5 What is the microbatch method? Crystallization in small drops under oil 100 + 100 nl to 1+1 µl

6. Douglas Instruments Microbatch seminar- slide 6 What is the microbatch method? Crystallization in small drops under oil 100 + 100 nl to 1+1 µl The oil prevents evaporation

7. Douglas Instruments Microbatch seminar- slide 7 Why is microbatch a good idea?

8. Douglas Instruments Microbatch seminar- slide 8 Why is microbatch a good idea? 1.Easy

9. Douglas Instruments Microbatch seminar- slide 9 Why is microbatch a good idea? 1.Easy 2.Gives better crystals in many cases – especially in screening

10. Douglas Instruments Microbatch seminar- slide 10 Why is microbatch a good idea? 1.Easy 2.Gives better crystals in many cases – especially in screening 3.It doesn’t matter if the security guard at the airport puts it through the x-ray machine upside down

11. Douglas Instruments Microbatch seminar- slide 11 Why is microbatch a good idea? 1.Easy 2.Gives better crystals in many cases – especially in screening 3.It doesn’t matter if the security guard at the airport puts it through the x-ray machine upside down 4.Cheap!

12. Douglas Instruments Microbatch seminar- slide 12 Microbatch c rystallization Volume of well - 12 microlitres

13. Douglas Instruments Microbatch seminar- slide 13 Microbatch c rystallization Volume of drop - 0.2 to 2 microlitres

14. Douglas Instruments Microbatch seminar- slide 14 Microbatch c rystallization (2-bore) microtip Oil Sample

15. Douglas Instruments Microbatch seminar- slide 15 Microbatch crystallization

16. Douglas Instruments Microbatch seminar- slide 16 Microbatch crystallization

17. Douglas Instruments Microbatch seminar- slide 17 Microbatch optimization – print out Row1 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row2 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row3 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row4 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010

18. Douglas Instruments Microbatch seminar- slide 18 Microbatch optimization – print out Row1 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row2 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row3 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010 Row4 50mg/ml BSA1.06 3M NaAc pH70.35 100% Pure green dye000000000000 95% PEG 600 dyed red0.120.110.10.080.070.060.050.040.030.020.010

19. Douglas Instruments Microbatch seminar- slide 19 ORYX 6 c rystallization system

20. Douglas Instruments Microbatch seminar- slide 20 Liquid-handling channel Motorized Hamilton gas-tight syringe (water) X 5 to microtip

21. Douglas Instruments Microbatch seminar- slide 21 ORYX 6 c rystallization system

22. Douglas Instruments Microbatch seminar- slide 22 Large-volume tip for oil

23. Douglas Instruments Microbatch seminar- slide 23 New Chassis design

24. Douglas Instruments Microbatch seminar- slide 24 New Chassis design

25. Douglas Instruments Microbatch seminar- slide 25 Left-hand tip: 2-bore Microtip – screening 5-bore Microtip – optimization

26. Douglas Instruments Microbatch seminar- slide 26 End of a 5-bore microtip 0.15 mm 0.9 mm

27. Douglas Instruments Microbatch seminar- slide 27 Sitting Drop

28. Douglas Instruments Microbatch seminar- slide 28 Sitting Drop - preparation Use a 2-bore microtip

29. Douglas Instruments Microbatch seminar- slide 29 Sitting Drop - preparation Use a 2-bore microtip Start with both bores full of water

30. Douglas Instruments Microbatch seminar- slide 30 Sitting Drop - preparation Air bubbles Use a 2-bore microtip Start with both bores full of water Suck up 1µl of air into both channels

31. Douglas Instruments Microbatch seminar- slide 31 Sitting Drop - preparation Air bubble Protein slug 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

32. Douglas Instruments Microbatch seminar- slide 32 Sitting Drop – dispensing cycle 1.Rinse in reservoir (1)

33. Douglas Instruments Microbatch seminar- slide 33 Sitting Drop – dispensing cycle 1.Rinse in reservoir 2.Move sideways and pick up clean solution (2) (1)

34. Douglas Instruments Microbatch seminar- slide 34 Sitting Drop – dispensing cycle 1.Rinse in reservoir 2.Move sideways and pick up clean solution 3.Dispense solution and protein (2) (1) (3)

35. Douglas Instruments Microbatch seminar- slide 35 Microbatch screening

36. Douglas Instruments Microbatch seminar- slide 36 Microbatch screening – dispensing cycle Screening solutions Target plate

37. Douglas Instruments Microbatch seminar- slide 37 Microbatch screening – dispensing cycle (1) 1.Pick up screening solution

38. Douglas Instruments Microbatch seminar- slide 38 Microbatch screening – dispensing cycle (2) (1) 1.Pick up screening solution 2.Transfer to microbatch drop

39. Douglas Instruments Microbatch seminar- slide 39 Microbatch screening – dispensing cycle (2) (1) + oil 1.Pick up screening solution 2.Transfer to microbatch drop; oil

40. Douglas Instruments Microbatch seminar- slide 40 Microbatch screening – dispensing cycle (2) (1) + oil (3) 1.Pick up screening solution 2.Transfer to microbatch drop; oil 3.Rinse

41. Douglas Instruments Microbatch seminar- slide 41 Microbatch optimization

42. Douglas Instruments Microbatch seminar- slide 42 Microbatch optimization – dispensing cycle (1) 1.Dispense five solutions together

43. Douglas Instruments Microbatch seminar- slide 43 Microbatch optimization – dispensing cycle (1) + oil 1.Dispense five solutions together 2.Oil

44. Douglas Instruments Microbatch seminar- slide 44 Microbatch optimization – dispensing cycle (1) + oil 1.Dispense five solutions together 2.Oil

45. Douglas Instruments Microbatch seminar- slide 45 Microbatch optimization – dispensing cycle (1) + oil 1.Dispense five solutions together 2.Oil

46. Douglas Instruments Microbatch seminar- slide 46 Central Composite design

47. Douglas Instruments Microbatch seminar- slide 47 Phase diagram of a protein [Protein] [Precipitant] clear precipitate

48. Douglas Instruments Microbatch seminar- slide 48 Phase diagram of a protein [Protein] [Precipitant] clear precipitate nucleation

49. Douglas Instruments Microbatch seminar- slide 49 Phase diagram of a protein [Protein] [Precipitant] clear precipitate nucleation metastable zone

50. Douglas Instruments Microbatch seminar- slide 50 Phase diagram of a protein [Protein] [Precipitant] c p n m.z. Vapor diffusion

51. Douglas Instruments Microbatch seminar- slide 51 Phase diagram of a protein [Protein] [Precipitant] c p n m.z. v.d. Microbatch

52. Douglas Instruments Microbatch seminar- slide 52 Phase diagram of a protein [Protein] [Precipitant] c p n m.z. v.d.. M.B.(paraffin) M.B.(par./si.)

53. Douglas Instruments Microbatch seminar- slide 53 Phase diagram of a protein [Protein] [Precipitant] p n m.z. v.d. M.B.(paraffin) OPTIMIZATION M.B.(par./si.) SCREENING

54. Douglas Instruments Microbatch seminar- slide 54 What % of protein should you use? [Protein] [Precipitant] n m.z. Microbatch with Si. / Par.: Precipitant saturated

55. Douglas Instruments Microbatch seminar- slide 55 What % of protein should you use? [Protein] [Precipitant] n m.z. Microbatch with Si. / Par.: Protein stock Precipitant stock Precipitant saturated 50%

56. Douglas Instruments Microbatch seminar- slide 56 What % of protein should you use? [Protein] [Precipitant] n m.z. Microbatch with Si. / Par.: Protein stock Precipitant stock Precipitant saturated 50% 66%

57. Douglas Instruments Microbatch seminar- slide 57 Screening: studies comparing microbatch with vapor diffusion ProteinsConditionsMBVD Extra hits for MB Extra hits for MB % Unique to MB Unique to VD 1996 Baldock et al. Douglas Ins. 648434125%1715 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.htmhttp://www.douglas.co.uk/rep2.htm

58. Douglas Instruments Microbatch seminar- slide 58 Screening: studies comparing microbatch with vapor diffusion ProteinsConditionsMBVD Extra hits for MB Extra hits for MB % Unique to MB Unique to VD 1996 Baldock et al. Douglas Ins. 648434125%1715 2000 D'Arcy et al. Hoffman -La Roche 1048104624268% 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.htmhttp://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.

59. Douglas Instruments Microbatch seminar- slide 59 Screening: studies comparing microbatch with vapor diffusion ProteinsConditionsMBVD Extra hits for MB Extra hits for MB % Unique to MB Unique to VD 1996 Baldock et al. Douglas Ins. 648434125%1715 2000 D'Arcy et al. Hoffman -La Roche 1048104624268% 2001 Noordeen et al. Novartis Pharma 848 - 576145153-8-5%95103 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.htmhttp://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. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt

60. Douglas Instruments Microbatch seminar- slide 60 Screening: studies comparing microbatch with vapor diffusion ProteinsConditionsMBVD Extra hits for MB Extra hits for MB % Unique to MB Unique to VD 1996 Baldock et al. Douglas Ins. 648434125%1715 2000 D'Arcy et al. Hoffman -La Roche 1048104624268% 2001 Noordeen et al. Novartis Pharma 848 - 576145153-8-5%95103 SugaharaSPring8 6288100841619% 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.htmhttp://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. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

61. Douglas Instruments Microbatch seminar- slide 61 Screening: studies comparing microbatch with vapor diffusion ProteinsConditionsMBVD Extra hits for MB Extra hits for MB % Unique to MB Unique to VD 1996 Baldock et al. Douglas Ins. 648434125%1715 2000 D'Arcy et al. Hoffman -La Roche 1048104624268% 2001 Noordeen et al. Novartis Pharma 848 - 576145153-8-5%95103 SugaharaSPring8 6288100841619% TOTAL 30 39234052 15% 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.htmhttp://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. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

62. Douglas Instruments Microbatch seminar- slide 62 OPTIMIZATION: about 50:50 In microbatch, there tends to be more precipitation initially; this may result in more nucleation

63. Douglas Instruments Microbatch seminar- slide 63 OPTIMIZATION: about 50:50 In microbatch, there tends to be more precipitation initially; this may result in more nucleation In a survey of about 30 protein samples at Imperial College, London, the best data was collected from MB in 50% of cases

64. Douglas Instruments Microbatch seminar- slide 64 OPTIMIZATION: about 50:50 In microbatch, there tends to be more precipitation initially; this may result in more nucleation In a survey of about 30 protein samples at Imperial College, London, the best data was collected from MB in 50% of cases Lesley Haire (NIMR, London) told me that out of 12 structures solved in the last few years, 5 relied on microbatch

65. Douglas Instruments Microbatch seminar- slide 65 OPTIMIZATION: about 50:50 From D’Arcy et al. A novel approach to crystallising proteins under oil. Journal of Crystal Growth 168 (1996) 175-180. Vapor diffusion Microbatch

66. Douglas Instruments Microbatch seminar- slide 66 Crystals obtained at 4ºC (Lesley Haire, Imperial College)

67. Douglas Instruments Microbatch seminar- slide 67 Crystals nucleated for 1 hr 4ºC, then grown at 18ºC

68. Douglas Instruments Microbatch seminar- slide 68 Case Study 2 Use of microseeding Yaakov Korkhin and Artem Evdokimov, Weizmann Institute of Science, Israel A newly isolated alcohol dehydrogenase from a thermophile was crystallized with PEG 4000, pH 5.5 - 8.6 VD crystals grew very rapidly and were poorly formed MB crystals were initially similar

69. Douglas Instruments Microbatch seminar- slide 69 [Protein] [Precipitant] p m.z. 1. Determination of phase diagram

70. Douglas Instruments Microbatch seminar- slide 70 A few good quality crystals were obtained

71. Douglas Instruments Microbatch seminar- slide 71 [Protein] [PEG 4K] Edge of nucleation – 16 % PEG

72. Douglas Instruments Microbatch seminar- slide 72 2. Microseeding was used 1.A well-formed crystal was broken up in 15.5% PEG 2.The mixture was spun 3.A series of dilutions was set up using the supernatant (1:1000 worked best)

73. Douglas Instruments Microbatch seminar- slide 73 [Protein] [PEG 4K] Reservoir – 16.5 % Droplet – 15.5 %

74. Douglas Instruments Microbatch seminar- slide 74 Reproducible good quality crystals were obtained with microseeding. Crystals diffracted to 2Å

75. Douglas Instruments Microbatch seminar- slide 75 Exactly the same conditions – but with no seeding solution - gave poor crystals

76. Douglas Instruments Microbatch seminar- slide 76 Vapor batch crystallization using volatile organic solvents Lesley Haire Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Winner of the competition for the Best Use of the Douglas Vapor Batch Plate First round - January 2005

77. Douglas Instruments Microbatch seminar- slide 77 Crystallisation of NTD of N-MLV capsid  Crystals were grown from hanging drops - Hampton Crystal Screen no.40, 20% PEG 4000, 20% v/v isopropanol, 0.1M Nacitrate pH 5.6 20mg/ml protein in the drops  Major problem - harvesting the crystals in the presence of isopropanol.  The crystals disintegrated as soon as the coverslip was opened.

78. Douglas Instruments Microbatch seminar- slide 78 Attempts to overcome the problem  Using sitting drops,  oil over the drops,  and handling crystals using constant humidity were only partially successful.  In microbatch experiments under oil, crystals were not stable and dissolved after a couple of days.  Crystals that were X-rayed had high mosaicity and could not be used for structure solution.

79. Douglas Instruments Microbatch seminar- slide 79 Vapor Batch trays (Douglas Instruments)

80. Douglas Instruments Microbatch seminar- slide 80 Procedure  Droplets (2  l) dispensed under a mixture of silicone/ paraffin oil using IMPAX 1-5 crystallisation robot.  A 6x4 spreadsheet was set up with XSTEP software varying; protein, 16-22 mg/ml; PEG 3350, 13-16%; all wells had 0.1M sodium citrate pH 5.6.  10% isopropanol was pipetted into the tray’s “moat” and the drops equilibrated overnight at 18  C.  Next day, the 10% isopropanol solution was replaced by 20% isopropanol

81. Douglas Instruments Microbatch seminar- slide 81 This method was used to grow crystals of NTD N-MLV capsid protein:  Crystals appeared after a couple of days.  Typically they were harvested and frozen after 10 days.  Crystals were very stable in drops for at least 6 months.  Diffraction to 1.9Å with low mosaicity.  Crystals did not grow in the controls without isopropanol in the moat. Capsid protein was provided by Nehar Mortuza

82. Douglas Instruments Microbatch seminar- slide 82 NTD N-tropic MLV- capsid protein G. B. Mortuza, L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye & I. A. Taylor. Nature (2004) 431 481-485.

83. Douglas Instruments Microbatch seminar- slide 83 Using “vapor batch” in screening Low ionic strength PEG screens Vary pH and PEG concentration +/- isopropanol or other volatile organic in the moat. High salt screens Use AmS04 or P04, set up duplicate trays, +/-10% isopropanol in the moat. The same principle could be used to test isopropanol or any other volatile additive with a selected screen dispensed in VB trays.

84. Douglas Instruments Microbatch seminar- slide 84 Crystals grown by VB with isopropanol 1918 H1 catalase Low ionic strength PEG screen, Sigma

85. Douglas Instruments Microbatch seminar- slide 85 Advantages of “vapor batch” cf. vapor diffusion:  Improved crystal stability  Easier crystal handling  Better diffraction from crystals grown under paraffin/silicone oil mixture.

86. Douglas Instruments Microbatch seminar- slide 86 Harvesting Crystals from Microbatch James Liu - University of Georgia Jeroen Mesters - University of Luebeck

87. Douglas Instruments Microbatch seminar- slide 87 Harvesting Crystals from Microbatch James Liu – B.C. Wang’s group, University of Georgia High-throughput crystallization for structural genomics

88. Douglas Instruments Microbatch seminar- slide 88 Harvesting Crystals from Microbatch James Liu Microbatch is easier because the oil prevents evaporation - you can work slowly! You can loop straight out of the droplet through the oil. James’ record – he mounted 98 crystals in one day!

89. Douglas Instruments Microbatch seminar- slide 89 Harvesting Crystals from Microbatch Digression: University of Georgia is unusual: it uses sitting drop for screening and microbatch for optimization. This reduces the solution volumes needed – and solutions can be reused many times.

90. Douglas Instruments Microbatch seminar- slide 90 Harvesting Crystals from Microbatch 1.Use a loop with a bent handle. 2.Make sure the crystal fits the loop well or the oil will drag it off. Jeroen Mesters:

91. Douglas Instruments Microbatch seminar- slide 91 Multivariate experimental design Almost all protein crystallization experiments have at least 4 parameters: 1.Protein concentration 2.Precipitant concentration 3.pH 4.Temperature 5.Additive ? …………….

92. Douglas Instruments Microbatch seminar- slide 92 Central Composite design

93. Douglas Instruments Microbatch seminar- slide 93 Box-Behnken design

94. Douglas Instruments Microbatch seminar- slide 94 The autodesign function of XSTEP ….

95. Douglas Instruments Microbatch seminar- slide 95 …. automatically fills a “spreadsheet” …

96. Douglas Instruments Microbatch seminar- slide 96 …. and XSTEP executes it.

97. Douglas Instruments Microbatch seminar- slide 97 The Biblical Zoo in Jerusalem

98. Douglas Instruments Microbatch seminar- slide 98 ORYX (arabian)

99. Douglas Instruments Microbatch seminar- slide 99 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, Vaughan Mills

100. Douglas Instruments Microbatch seminar- slide 100 And finally - Yaakov showed me …

101. Douglas Instruments Microbatch seminar- slide 101 What Yaakov saw

102. Douglas Instruments Microbatch seminar- slide 102 How can we do vapor diffusion as easily as microbatch?

103. Douglas Instruments Microbatch seminar- slide 103 Vapor Diffusion effect demonstrated by increasing reservoir concentration Days Number of crystals  0.5 M AS  1.0 M AS

104. Douglas Instruments Microbatch seminar- slide 104 Experimental Design Steps Step 1. “Primary Screen.” Approx. 30-dimensional search. E.g. Sparse Matrix or Incomplete Factorial Step 2. “Targeted Screen” Approx. 10-dimensional search. E.g. Incomplete factorial or Crystool™ optimization Step 3. “Multidimensional Grid” Approx. 4-dimensional search. E.g. Central Composite, Box Behnken - XSTEP Autodesign Step 4. “2-D Grid” Approx. 2-dimensional search. E.g. XSTEP grids.