A4

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
• 100 100 nl to 11 µl

6
What is the microbatch method?

• Crystallization in small drops under oil
• 100 100 nl to 11 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 doesnt 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 doesnt 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
18
Microbatch optimization print out
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

23
Phase diagram of a protein

p
n
Protein
m.z.
Vapor diffusion
c
Precipitant
24
Phase diagram of a protein

p
n
Microbatch
Protein
m.z.
v.d.
c
Precipitant
25
Phase diagram of a protein

p
n
M.B.(paraffin)
Protein
m.z.
v.d..
M.B.(par./si.)
c
Precipitant
26
Phase diagram of a protein

p
n
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

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
32
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. DArcy,
G.E. Dale, M. Stihle, B. DArcy. Results
reported at the 8th International Conference on
the Crystallization of Biological Macromolecules,
May 18, 2000.
33
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. DArcy,
G.E. Dale, M. Stihle, B. DArcy. 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
34
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. DArcy,
G.E. Dale, M. Stihle, B. DArcy. 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.
35
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. DArcy,
G.E. Dale, M. Stihle, B. DArcy. 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.
36
OPTIMIZATION about 5050

• In microbatch, there tends to be more
  precipitation initially this may result in more
  nucleation

37
OPTIMIZATION about 5050

• 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 5050

• 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 5050
Vapor diffusion
Microbatch

From DArcy et al. A novel approach to
crystallising proteins under oil. Journal of
Crystal Growth 168 (1996) 175-180.
40

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

41
Case Study 2Use 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

42
Reservoir 16.5
Droplet 15.5
Protein
PEG 4K
43
Reproducible good quality crystals wereobtained
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) 431 481-485.
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.
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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.