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. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

3. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

4. What is the microbatch method?
Crystallization in small drops under oil

5. What is the microbatch method?
Crystallization in small drops under oil
nl to 1+1 µl

6. What is the microbatch method?
Crystallization in small drops under oil
nl to 1+1 µl
The oil prevents evaporation

7. Why is microbatch a good idea?

8. Why is microbatch a good idea?
Easy

9. Why is microbatch a good idea?
Easy
Gives better crystals in many cases – especially in screening

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

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

12. Microbatch crystallization
Volume of well -
12 microlitres

13. Microbatch crystallization
Volume of drop -
0.2 to 2 microlitres

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

15. Microbatch crystallization

16. Microbatch crystallization

17. Microbatch optimization – print out
Row 1 50
mg/ml BSA
1.06 3
M NaAc pH7
0.35
100
% Pure green dye 95
% PEG 600 dyed red
0.12
0.11
0.1
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01 2 4

18. Microbatch optimization – print out
Row 1 50
mg/ml BSA
1.06 3
M NaAc pH7
0.35
100
% Pure green dye 95
% PEG 600 dyed red
0.12
0.11
0.1
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01 2 4

19. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

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

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

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

23. Phase diagram of a protein
m.z.
Vapor diffusion c
[Precipitant]

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

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

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

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

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

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

30. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

31. Screening: studies comparing microbatch with vapor diffusion
Proteins
Conditions MB VD
Extra hits for MB  
Extra hits for MB %
Unique to MB
Unique to VD
1996
Baldock et al.
Douglas Ins. 6 48 43 41 2 5% 17 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 or:

32. Screening: studies comparing microbatch with vapor diffusion
Proteins
Conditions MB VD
Extra hits for MB  
Extra hits for MB %
Unique to MB
Unique to VD
1996
Baldock et al.
Douglas Ins. 6 48 43 41 2 5% 17 15
2000
D'Arcy
et al.
Hoffman-La Roche 10
104 62 42
68%
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 or:
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.

33. Screening: studies comparing microbatch with vapor diffusion
Proteins
Conditions MB VD
Extra hits for MB  
Extra hits for MB %
Unique to MB
Unique to VD
1996
Baldock et al.
Douglas Ins. 6 48 43 41 2 5% 17 15
2000
D'Arcy
et al.
Hoffman-La Roche 10
104 62 42
68%
2001
Noordeen et al.
Novartis Pharma 8
145
153 -8
-5% 95
103
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 or:
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.

34. Screening: studies comparing microbatch with vapor diffusion
Proteins
Conditions MB VD
Extra hits for MB  
Extra hits for MB %
Unique to MB
Unique to VD
1996
Baldock et al.
Douglas Ins. 6 48 43 41 2 5% 17 15
2000
D'Arcy
et al.
Hoffman-La Roche 10
104 62 42
68%
2001
Noordeen et al.
Novartis Pharma 8
145
153 -8
-5% 95
103
Sugahara
SPring8
288
100 84 16
19%
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 or:
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.
Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

35. Screening: studies comparing microbatch with vapor diffusion
Proteins
Conditions MB VD
Extra hits for MB  
Extra hits for MB %
Unique to MB
Unique to VD
1996
Baldock et al.
Douglas Ins. 6 48 43 41 2 5% 17 15
2000
D'Arcy
et al.
Hoffman-La Roche 10
104 62 42
68%
2001
Noordeen et al.
Novartis Pharma 8
145
153 -8
-5% 95
103
Sugahara
SPring8
288
100 84 16
19%
TOTAL
 30
392
340
52 
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 or:
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.
Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

36. OPTIMIZATION: about 50:50
In microbatch, there tends to be more precipitation initially; this may result in more nucleation

37. 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

38. 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

39. OPTIMIZATION: about 50:50
Vapor
diffusion
Microbatch
From D’Arcy et al. A novel approach to crystallising proteins under oil. Journal of Crystal Growth 168 (1996)

40. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

41. 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
VD crystals grew very rapidly and were poorly formed
MB crystals were initially similar

42. Droplet – 15.5 %
Reservoir – 16.5 %
[Protein]
[PEG 4K]

43. Reproducible good quality crystals were obtained with microseeding
Reproducible good quality crystals were obtained with microseeding. Crystals diffracted to 2Å

44. Vapor Batch trays (Douglas Instruments)

45. 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)

46. Crystals obtained at 4ºC (Lesley Haire, Imperial College)

47. Crystals nucleated for 1 hr 4ºC, then grown at 18ºC

48. What is the microbatch method?
Phase diagrams
Comparisons of microbatch and vapor diffusion
Case studies
Experimental design

49. Multivariate experimental design
Almost all protein crystallization experiments have at least 4 parameters:
Protein concentration
Precipitant concentration pH
Temperature
Additive ? …………….

50. Central Composite design

51. Box-Behnken design

52. The autodesign function of XSTEP ….

53. …. automatically fills a “spreadsheet” …

54. …. and XSTEP executes it.

55. ORYX (arabian)

56. 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.