Patrick Shaw Stewart Imperial College London Professor David

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 Microbatch seminar- slide 1 Douglas Instruments Should we be doing more crystallization by the microbatch method?

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

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

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

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

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

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

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

1. Easy 2. Gives better crystals in many cases – especially in screening Microbatch seminar- slide 9 Douglas Instruments 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 Microbatch seminar- slide 10 Douglas Instruments 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! Microbatch seminar- slide 11 Douglas Instruments Why is microbatch a good idea?

Volume of well 12 microlitres Microbatch seminar- slide 12 Douglas Instruments Microbatch crystallization

Volume of drop 0. 2 to 2 microlitres Microbatch seminar- slide 13 Douglas Instruments Microbatch crystallization

(2 -bore) microtip Oil Sample Microbatch seminar- slide 14 Douglas Instruments Microbatch crystallization

Microbatch seminar- slide 15 Douglas Instruments Microbatch crystallization

Microbatch seminar- slide 16 Douglas Instruments Microbatch crystallization

Row 1 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 2 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 3 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 4 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Microbatch seminar- slide 17 Douglas Instruments Microbatch optimization – print out

Row 1 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 2 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 3 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Row 4 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure green dye 95% PEG 600 dyed red 1. 06 0. 35 0 0 0. 12 0. 11 1. 06 0. 35 0 0. 08 1. 06 0. 35 0 0. 07 1. 06 0. 35 0 0. 06 1. 06 0. 35 0 0. 05 1. 06 0. 35 0 0. 04 1. 06 0. 35 0 0. 03 1. 06 0. 35 0 0. 02 1. 06 0. 35 0 0. 01 1. 06 0. 35 0 0 Microbatch seminar- slide 18 Douglas Instruments Microbatch optimization – print out

2. Phase diagrams 3. Comparisons of microbatch and vapor diffusion 4. Case studies 5. Experimental design Microbatch seminar- slide 19 Douglas Instruments 1. What is the microbatch method?

precipitate [Protein] clear [Precipitant] Microbatch seminar- slide 20 Douglas Instruments Phase diagram of a protein

precipitate nucleation [Protein] clear [Precipitant] Microbatch seminar- slide 21 Douglas Instruments Phase diagram of a protein

precipitate nucleation [Protein] metastable zone clear [Precipitant] Microbatch seminar- slide 22 Douglas Instruments Phase diagram of a protein

p n [Protein] Vapor diffusion c [Precipitant] Microbatch seminar- slide 23 m. z. Douglas Instruments Phase diagram of a protein

p n Microbatch [Protein] m. z. v. d. c [Precipitant] Microbatch seminar- slide 24 Douglas Instruments Phase diagram of a protein

p n M. B. (paraffin) [Protein] m. z. v. d. . M. B. (par. /si. ) c [Precipitant] Microbatch seminar- slide 25 Douglas Instruments Phase diagram of a protein

p n M. B. (paraffin) OPTIMIZATION [Protein] m. z. v. d. M. B. (par. /si. ) SCREENING [Precipitant] Microbatch seminar- slide 26 Douglas Instruments Phase diagram of a protein

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

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

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

2. Phase diagrams 3. Comparisons of microbatch and vapor diffusion 4. Case studies 5. Experimental design Microbatch seminar- slide 30 Douglas Instruments 1. What is the microbatch method?

1996 Baldock et al. Proteins Conditions MB VD Douglas Ins. 6 48 43 41 Extra hits Unique to for MB % MB VD 2 5% 17 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/rep 2. htm Microbatch seminar- slide 31 15 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

Extra hits Unique to for MB % MB VD Proteins Conditions MB VD 1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman-La Roche 10 48 104 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/rep 2. htm A. D’Arcy, G. E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8 th International Conference on the Crystallization of Biological Macromolecules, May 18, 2000. Microbatch seminar- slide 32 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

Extra hits Unique to for MB % MB VD Proteins Conditions MB VD 1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman-La Roche 10 48 104 62 42 68% 8 48 - 576 145 153 -8 -5% 95 103 2001 Novartis Noordeen et al. Pharma 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/rep 2. htm A. D’Arcy, G. E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8 th 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/P 6. ppt Microbatch seminar- slide 33 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

Extra hits Unique to for MB % MB VD Proteins Conditions MB VD 1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman-La Roche 10 48 104 62 42 68% Novartis Noordeen et al. Pharma 8 48 - 576 145 153 -8 -5% 95 103 Sugahara 6 288 100 84 16 19% 2001 SPring 8 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/rep 2. htm A. D’Arcy, G. E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8 th 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/P 6. ppt Misuaki Sugahara, Riken Harima Institute, SPring 8. Personal communication. Microbatch seminar- slide 34 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

Extra hits Unique to for MB % MB VD Proteins Conditions MB VD 1996 Baldock et al. Douglas Ins. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman-La Roche 10 48 104 62 42 68% Novartis Noordeen et al. Pharma 8 48 - 576 145 153 -8 -5% 95 103 Sugahara SPring 8 6 288 100 84 16 19% TOTAL 30 392 340 52 15% 2001 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/rep 2. htm A. D’Arcy, G. E. Dale, M. Stihle, B. D’Arcy. Results reported at the 8 th 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/P 6. ppt Misuaki Sugahara, Riken Harima Institute, SPring 8. Personal communication. Microbatch seminar- slide 35 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

• In microbatch, there tends to be more precipitation initially; this may result in more nucleation Microbatch seminar- slide 36 Douglas Instruments 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 Microbatch seminar- slide 37 Douglas Instruments 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 Microbatch seminar- slide 38 Douglas Instruments OPTIMIZATION: about 50: 50

Vapor diffusion From D’Arcy et al. A novel approach to crystallising proteins under oil. Journal of Crystal Growth 168 (1996) 175 -180. Microbatch seminar- slide 39 Microbatch Douglas Instruments OPTIMIZATION: about 50: 50

2. Phase diagrams 3. Comparisons of microbatch and vapor diffusion 4. Case studies 5. Experimental design Microbatch seminar- slide 40 Douglas Instruments 1. What is the microbatch method?

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, p. H 5. 5 - 8. 6 • VD crystals grew very rapidly and were poorly formed Microbatch seminar- slide 41 • MB crystals were initially similar Douglas Instruments Case Study 2

[Protein] [PEG 4 K] Microbatch seminar- slide 42 Douglas Instruments Reservoir – 16. 5 % Droplet – 15. 5 %

Microbatch seminar- slide 43 Douglas Instruments Reproducible good quality crystals were obtained with microseeding. Crystals diffracted to 2Å

(Douglas Instruments) Microbatch seminar- slide 44 Douglas Instruments Vapor Batch trays

G. B. Mortuza, L. F. Haire, A. Stevens, S. J. Smerdon, J. P. Stoye & I. A. Taylor. Nature (2004) 431 481 -485. Microbatch seminar- slide 45 Douglas Instruments NTD N-tropic MLV- capsid protein

(Lesley Haire, Imperial College) Microbatch seminar- slide 46 Douglas Instruments Crystals obtained at 4ºC

Microbatch seminar- slide 47 Douglas Instruments Crystals nucleated for 1 hr 4ºC, then grown at 18ºC

2. Phase diagrams 3. Comparisons of microbatch and vapor diffusion 4. Case studies 5. Experimental design Microbatch seminar- slide 48 Douglas Instruments 1. What is the microbatch method?

Almost all protein crystallization experiments have at least 4 parameters: Microbatch seminar- slide 49 1. Protein concentration 2. Precipitant concentration 3. p. H 4. Temperature 5. Additive ? ……………. Douglas Instruments Multivariate experimental design

Microbatch seminar- slide 50 Douglas Instruments Central Composite design

Microbatch seminar- slide 51 Douglas Instruments Box-Behnken design

Microbatch seminar- slide 52 Douglas Instruments The autodesign function of XSTEP ….

Microbatch seminar- slide 53 Douglas Instruments …. automatically fills a “spreadsheet” …

Microbatch seminar- slide 54 Douglas Instruments …. and XSTEP executes it.

Microbatch seminar- slide 55 Douglas Instruments ORYX (arabian)

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. Microbatch seminar- slide 56 Douglas Instruments Experimental Design Steps