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

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

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

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

What is the microbatch method? Crystallization in small drops under oil 100 + 100 nl to 1+1 µl

What is the microbatch method? Crystallization in small drops under oil 100 + 100 nl to 1+1 µl The oil prevents evaporation

Why is microbatch a good idea?

Why is microbatch a good idea? Easy

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

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

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!

Microbatch crystallization Volume of well - 12 microlitres

Microbatch crystallization Volume of drop - 0.2 to 2 microlitres

Microbatch crystallization (2-bore) microtip Oil Sample

Microbatch crystallization

Microbatch crystallization

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 170-174 or: http://www.douglas.co.uk/rep2.htm

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 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.

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 48 - 576 145 153 -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 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. N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt

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 48 - 576 145 153 -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 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. 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.

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 48 - 576 145 153 -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 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. 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.

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

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

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

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) 175-180.

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

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

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

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

Vapor Batch trays (Douglas Instruments)

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.

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

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

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

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

Central Composite design

Box-Behnken design

The autodesign function of XSTEP ….

…. automatically fills a “spreadsheet” …

…. and XSTEP executes it.

ORYX (arabian)

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.