Скачать презентацию microbatch method Patrick Shaw Stewart Imperial College London Скачать презентацию microbatch method Patrick Shaw Stewart Imperial College London

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microbatch method? Patrick Shaw Stewart Imperial College, London: Professor David M. Blow, Patrick Shaw 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, James Smith, Richard Briggs Microbatch seminar- slide 1 Douglas Instruments Should we be doing more crystallization by the

 • Crystallization in small drops under oil Microbatch seminar- slide 2 Douglas Instruments • Crystallization in small drops under oil Microbatch seminar- slide 2 Douglas Instruments What is microbatch crystallization?

 (2 -bore) microtip Oil Sample Microbatch seminar- slide 3 Douglas Instruments Microbatch crystallization (2 -bore) microtip Oil Sample Microbatch seminar- slide 3 Douglas Instruments Microbatch crystallization

 Microbatch seminar- slide 4 Douglas Instruments Microbatch crystallization Microbatch seminar- slide 4 Douglas Instruments Microbatch crystallization

 Microbatch seminar- slide 5 Douglas Instruments Microbatch crystallization Microbatch seminar- slide 5 Douglas Instruments Microbatch crystallization

 0. 15 mm 0. 9 mm Microbatch seminar- slide 6 Douglas Instruments End 0. 15 mm 0. 9 mm Microbatch seminar- slide 6 Douglas Instruments End of a 5 -bore microtip

Row 1 50 mg/ml BSA 3 M Na. Ac p. H 7 100% Pure 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. 12 1. 06 0. 35 0 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. 12 1. 06 0. 35 0 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. 12 1. 06 0. 35 0 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. 12 1. 06 0. 35 0 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 7 Douglas Instruments Microbatch optimization – print out

Microbatch seminar- slide 8 Douglas Instruments ORYX 6 crystallization system Microbatch seminar- slide 8 Douglas Instruments ORYX 6 crystallization system

 • Versatile – optimization, additives etc. Microbatch seminar- slide 9 Douglas Instruments Oryx • Versatile – optimization, additives etc. Microbatch seminar- slide 9 Douglas Instruments Oryx Crystallization System

 • Versatile – optimization, additives etc. • Wastes very little protein (around 0. • Versatile – optimization, additives etc. • Wastes very little protein (around 0. 5 µl per experiment) Microbatch seminar- slide 10 Douglas Instruments Oryx Crystallization System

 • Sitting drops Microbatch seminar- slide 11 – 0. 1+0. 1 to 1+1µ • Sitting drops Microbatch seminar- slide 11 – 0. 1+0. 1 to 1+1µ Douglas Instruments Oryx Crystallization System

 • Sitting drops – 0. 1+0. 1 to 1+1µ • Microbatch screening – • Sitting drops – 0. 1+0. 1 to 1+1µ • Microbatch screening – 0. 1+0. 1 to 1+1µl Microbatch seminar- slide 12 Douglas Instruments Oryx Crystallization System

 • Sitting drops – 0. 1+0. 1 to 1+1µ • Microbatch screening – • Sitting drops – 0. 1+0. 1 to 1+1µ • Microbatch screening – 0. 1+0. 1 to 1+1µl • Microbatch optimization – 0. 5+0. 5 to 1+1µl Microbatch seminar- slide 13 Douglas Instruments Oryx Crystallization System

2 -bore Microtip – screening 5 -bore Microtip – optimization Microbatch seminar- slide 14 2 -bore Microtip – screening 5 -bore Microtip – optimization Microbatch seminar- slide 14 Douglas Instruments How does Oryx work?

Microbatch seminar- slide 15 Douglas Instruments Sitting Drop Microbatch seminar- slide 15 Douglas Instruments Sitting Drop

 • Use a 2 -bore microtip Microbatch seminar- slide 16 Douglas Instruments Sitting • Use a 2 -bore microtip Microbatch seminar- slide 16 Douglas Instruments Sitting Drop - preparation

 • • Use a 2 -bore microtip Start with bores full of water • • Use a 2 -bore microtip Start with bores full of water Microbatch seminar- slide 17 Douglas Instruments Sitting Drop - preparation

 • Douglas Instruments Sitting Drop - preparation Use a 2 -bore microtip • • Douglas Instruments Sitting Drop - preparation Use a 2 -bore microtip • Start with bores full of water • Suck up 1µl of air into both channels Air bubbles Microbatch seminar- slide 18

Air bubble • Use a 2 -bore microtip • Start with bores full of Air bubble • Use a 2 -bore microtip • Start with bores full of water • Suck up 1µl of air into both channels • Suck up protein required for experiment + 0. 25 µl Microbatch seminar- slide 19 Protein slug Douglas Instruments Sitting Drop - preparation

 1. Rinse in reservoir (1) Microbatch seminar- slide 20 Douglas Instruments Sitting Drop 1. Rinse in reservoir (1) Microbatch seminar- slide 20 Douglas Instruments Sitting Drop – dispensing cycle

 1. Rinse in reservoir 2. Move sideways and pick up clean solution (2) 1. Rinse in reservoir 2. Move sideways and pick up clean solution (2) Microbatch seminar- slide 21 (1) Douglas Instruments Sitting Drop – dispensing cycle

 1. Rinse in reservoir 2. Move sideways and pick up clean solution (3) 1. Rinse in reservoir 2. Move sideways and pick up clean solution (3) 3. Dispense solution and protein (2) Microbatch seminar- slide 22 (1) Douglas Instruments Sitting Drop – dispensing cycle

Microbatch seminar- slide 23 Douglas Instruments Hardware Microbatch seminar- slide 23 Douglas Instruments Hardware

 Microbatch seminar- slide 24 Douglas Instruments IMPAX 1 -5 crystallization system Microbatch seminar- slide 24 Douglas Instruments IMPAX 1 -5 crystallization system

Motorized Hamilton gas-tight syringe (water) Ground-glass syringe (water) Valve Disposable syringe (reagents) Valve Microtip Motorized Hamilton gas-tight syringe (water) Ground-glass syringe (water) Valve Disposable syringe (reagents) Valve Microtip Microbatch seminar- slide 25 X 5 Douglas Instruments Liquid-handling channel

 Microbatch seminar- slide 26 Douglas Instruments IMPAX 1 -5 crystallization system Microbatch seminar- slide 26 Douglas Instruments IMPAX 1 -5 crystallization system

 Microbatch seminar- slide 27 Douglas Instruments ORYX 6 crystallization system Microbatch seminar- slide 27 Douglas Instruments ORYX 6 crystallization system

Microbatch seminar- slide 28 Douglas Instruments ORYX 6 crystallization system Microbatch seminar- slide 28 Douglas Instruments ORYX 6 crystallization system

Microbatch seminar- slide 29 Douglas Instruments Microbatch screening Microbatch seminar- slide 29 Douglas Instruments Microbatch screening

Target plate Screening solutions Microbatch seminar- slide 30 Douglas Instruments Microbatch screening – dispensing Target plate Screening solutions Microbatch seminar- slide 30 Douglas Instruments Microbatch screening – dispensing cycle

1. Pick up screening solution Microbatch seminar- slide 31 (1) Douglas Instruments Microbatch screening 1. Pick up screening solution Microbatch seminar- slide 31 (1) Douglas Instruments Microbatch screening – dispensing cycle

1. Pick up screening solution 2. Transfer to microbatch drop Microbatch seminar- slide 32 1. Pick up screening solution 2. Transfer to microbatch drop Microbatch seminar- slide 32 (2) (1) Douglas Instruments Microbatch screening – dispensing cycle

1. Pick up screening (2) solution + oil 2. Transfer to microbatch drop; oil 1. Pick up screening (2) solution + oil 2. Transfer to microbatch drop; oil Microbatch seminar- slide 33 (1) Douglas Instruments Microbatch screening – dispensing cycle

1. Pick up screening (2) solution + oil 2. Transfer to microbatch drop; oil 1. Pick up screening (2) solution + oil 2. Transfer to microbatch drop; oil 3. Rinse Microbatch seminar- slide 34 (1) (3) Douglas Instruments Microbatch screening – dispensing cycle

Microbatch seminar- slide 35 Douglas Instruments Microbatch optimization Microbatch seminar- slide 35 Douglas Instruments Microbatch optimization

1. Dispense five solutions together Microbatch seminar- slide 36 (1) Douglas Instruments Microbatch optimization 1. Dispense five solutions together Microbatch seminar- slide 36 (1) Douglas Instruments Microbatch optimization – dispensing cycle

1. Dispense five solutions together 2. Oil Microbatch seminar- slide 37 (1) + oil 1. Dispense five solutions together 2. Oil Microbatch seminar- slide 37 (1) + oil Douglas Instruments Microbatch optimization – dispensing cycle

1. Dispense five solutions together 2. Oil Microbatch seminar- slide 38 (1) + oil 1. Dispense five solutions together 2. Oil Microbatch seminar- slide 38 (1) + oil Douglas Instruments Microbatch optimization – dispensing cycle

1. Dispense five solutions together 2. Oil Microbatch seminar- slide 39 (1) + oil 1. Dispense five solutions together 2. Oil Microbatch seminar- slide 39 (1) + oil Douglas Instruments Microbatch optimization – dispensing cycle

 precipitate [Protein] clear [Precipitant] Microbatch seminar- slide 40 Douglas Instruments Phase diagram of precipitate [Protein] clear [Precipitant] Microbatch seminar- slide 40 Douglas Instruments Phase diagram of a protein

 precipitate nucleation [Protein] clear [Precipitant] Microbatch seminar- slide 41 Douglas Instruments Phase diagram precipitate nucleation [Protein] clear [Precipitant] Microbatch seminar- slide 41 Douglas Instruments Phase diagram of a protein

 precipitate nucleation [Protein] metastable zone clear [Precipitant] Microbatch seminar- slide 42 Douglas Instruments precipitate nucleation [Protein] metastable zone clear [Precipitant] Microbatch seminar- slide 42 Douglas Instruments Phase diagram of a protein

 p n [Protein] c Vapor diffusion [Precipitant] Microbatch seminar- slide 43 m. z. p n [Protein] c Vapor diffusion [Precipitant] Microbatch seminar- slide 43 m. z. Douglas Instruments Phase diagram of a protein

 p n Microbatch [Protein] m. z. v. d. c [Precipitant] Microbatch seminar- slide p n Microbatch [Protein] m. z. v. d. c [Precipitant] Microbatch seminar- slide 44 Douglas Instruments Phase diagram of a protein

 p n M. B. (paraffin) [Protein] m. z. v. d. . M. B. p n M. B. (paraffin) [Protein] m. z. v. d. . M. B. (par. /si. ) c [Precipitant] Microbatch seminar- slide 45 Douglas Instruments Phase diagram of a protein

 p n M. B. (paraffin) OPTIMIZATION [Protein] m. z. v. d. M. B. p n M. B. (paraffin) OPTIMIZATION [Protein] m. z. v. d. M. B. (par. /si. ) SCREENING [Precipitant] Microbatch seminar- slide 46 Douglas Instruments Phase diagram of a protein

Microbatch with Si. / Par. : [Protein] n Protein stock m. z. 50% [Precipitant] Microbatch with Si. / Par. : [Protein] n Protein stock m. z. 50% [Precipitant] Microbatch seminar- slide 47 Precipitant saturated Precipitant stock Douglas Instruments What % of protein should you use?

Microbatch with Si. / Par. : [Protein] n Protein stock m. z. 66% 50% Microbatch with Si. / Par. : [Protein] n Protein stock m. z. 66% 50% [Precipitant] Microbatch seminar- slide 48 Precipitant saturated Precipitant stock Douglas Instruments What % of protein should you use?

 Baldock et Douglas Ins. 1996 al. Proteins Conditions 6 48 MB VD 43 Baldock et Douglas Ins. 1996 al. Proteins Conditions 6 48 MB VD 43 41 Extra hits for Unique MB % to MB to VD 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/rep 2. htm Microbatch seminar- slide 49 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

 Proteins Conditions MB VD Extra hits for Unique MB % to MB to Proteins Conditions MB VD Extra hits for Unique MB % to MB to VD Baldock et Douglas Ins. 1996 al. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. 10 48 104 62 42 68% Hoffman. La Roche 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 50 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

 Proteins Conditions MB VD Extra hits for Unique MB % to MB to Proteins Conditions MB VD Extra hits for Unique MB % to MB to VD Baldock et Douglas Ins. 1996 al. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman. La Roche 10 48 104 62 42 68% Noordeen Novartis Pharma 2001 et al. 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/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 51 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

 Proteins Conditions MB VD Extra hits for Unique MB % to MB to Proteins Conditions MB VD Extra hits for Unique MB % to MB to VD Baldock et Douglas Ins. 1996 al. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman. La Roche 10 48 104 62 42 68% Noordeen Novartis Pharma 2001 et al. 8 48 - 576 145 153 -8 -5% 95 103 6 288 100 84 16 19% Sugahara 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 52 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

 Proteins Conditions MB VD Extra hits for Unique MB % to MB to Proteins Conditions MB VD Extra hits for Unique MB % to MB to VD Baldock et Douglas Ins. 1996 al. 6 48 43 41 2 5% 17 15 D'Arcy 2000 et al. Hoffman. La Roche 10 48 104 62 42 68% Noordeen Novartis Pharma 2001 et al. 8 48 - 576 145 153 -8 -5% 95 103 6 288 100 84 16 19% 30 52 15% Sugahara SPring 8 TOTAL 392 340 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 53 Douglas Instruments Screening: studies comparing microbatch with vapor diffusion

 • In microbatch, there tends to be more precipitation initially; this may result • In microbatch, there tends to be more precipitation initially; this may result in more nucleation • In a survey of about 30 proteins at Imperial College, London, the best data was collected from MB in about 50% of cases Microbatch seminar- slide 54 Douglas Instruments OPTIMIZATION: about 50: 50

Vapor diffusion From D’Arcy et al. A novel approach to crystallising proteins under oil. 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 55 Microbatch Douglas Instruments OPTIMIZATION: about 50: 50

 Elena Conti, Imperial College, London Recombinant firefly luciferase • VD crystals grown at Elena Conti, Imperial College, London Recombinant firefly luciferase • VD crystals grown at 4ºC tended to redissolve during observation at 18ºC • MB crystals (shown) were stable at all temperatures Microbatch seminar- slide 56 Douglas Instruments Case Study 1

Use of microseeding Yaakov Korkhin and Artem Evdokimov, Weizmann Institute of Science, Israel A 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 57 • MB crystals were initially similar Douglas Instruments Case Study 2

 p [Protein] m. z. [Precipitant] Microbatch seminar- slide 58 Douglas Instruments 1. Determination p [Protein] m. z. [Precipitant] Microbatch seminar- slide 58 Douglas Instruments 1. Determination of phase diagram

 Microbatch seminar- slide 59 Douglas Instruments A few good quality crystals were obtained Microbatch seminar- slide 59 Douglas Instruments A few good quality crystals were obtained

1. The edge of the nucleation zone was identified (16% PEG) 2. A well-formed 1. The edge of the nucleation zone was identified (16% PEG) 2. A well-formed crystal was broken up in 15. 5% PEG 3. The mixture was spun 4. A series of dilutions was set up using the supernatant (1: 1000 worked best) 5. VD reservoirs were set up just inside the nucleation zone (16. 5% PEG) 6. Sitting drops were set up just outside the nucleation zone (15. 5% PEG) 7. 0. 2 µl of seeding dilution was added to each 2 µl sitting drop. Microbatch seminar- slide 60 Douglas Instruments 2. Microseeding was used

Reservoir – 16. 5 % [Protein] [PEG 4 K] Microbatch seminar- slide 61 Douglas Reservoir – 16. 5 % [Protein] [PEG 4 K] Microbatch seminar- slide 61 Douglas Instruments Droplet – 15. 5 %

 Microbatch seminar- slide 62 Douglas Instruments Reproducible good quality crystals were obtained with Microbatch seminar- slide 62 Douglas Instruments Reproducible good quality crystals were obtained with microseeding. Crystals diffracted to 2Å

 Microbatch seminar- slide 63 Douglas Instruments Exactly the same conditions – but with Microbatch seminar- slide 63 Douglas Instruments Exactly the same conditions – but with no seeding solution - gave poor crystals

(Lesley Haire, Imperial College) Microbatch seminar- slide 64 Douglas Instruments Crystals obtained at 4ºC (Lesley Haire, Imperial College) Microbatch seminar- slide 64 Douglas Instruments Crystals obtained at 4ºC

 Microbatch seminar- slide 65 Douglas Instruments Crystals nucleated for 1 hr 4ºC, then Microbatch seminar- slide 65 Douglas Instruments Crystals nucleated for 1 hr 4ºC, then grown at 18ºC

James Liu - University of Georgia Jeroen Mesters - University of Luebeck Microbatch seminar- James Liu - University of Georgia Jeroen Mesters - University of Luebeck Microbatch seminar- slide 66 Douglas Instruments Harvesting Crystals from Microbatch

James Liu High-throughput crystallization for structural genomics University of Georgia is unusual: it uses James Liu High-throughput crystallization for structural genomics University of Georgia is unusual: it uses sitting drop for screening and microbatch for optimization. (This reduces the solution volumes needed – solutions can be reused. ) Also, harvesting from microbatch is easier! Microbatch seminar- slide 67 Douglas Instruments Harvesting Crystals from Microbatch

James Liu 1. Microbatch is easier because the oil prevents evaporation; you can work James Liu 1. Microbatch is easier because the oil prevents evaporation; you can work slowly! 2. You can loop straight out of the droplet through the oil. 3. James’ record – he mounted 98 crystals mounted in one day! Microbatch seminar- slide 68 Douglas Instruments Harvesting Crystals from Microbatch

Jeroen Mesters: 1. Use a loop with a bent handle. 2. Make sure the Jeroen Mesters: 1. Use a loop with a bent handle. 2. Make sure the crystal fits the loop well or the oil will drag it off. Microbatch seminar- slide 69 Douglas Instruments Harvesting Crystals from Microbatch

Jeroen Mesters: For cryo data collection, don’t use regular cryoprotectant: drag crystal on loop Jeroen Mesters: For cryo data collection, don’t use regular cryoprotectant: drag crystal on loop through dry paraffin oil to remove mother liquor. (Make batches of dry oil in Eppendorf tubes using Speedvac overnight. Store frozen. Discard a few days after opening. ) Microbatch seminar- slide 70 Douglas Instruments Harvesting Crystals from Microbatch

Heavy atom derivatization: 1. Make a stock solution with the same concentration of precipitant Heavy atom derivatization: 1. Make a stock solution with the same concentration of precipitant as the drop, plus 1 to 10 m. M of the heavy atom. 2. Add 0. 2 µl of heavy atom solution to droplet. Add far from the crystal. Leave 24 hours. 3. Loop out in the normal way. Microbatch seminar- slide 71 Douglas Instruments Harvesting Crystals from Microbatch

If you can’t freeze your crystals: 1. You need to develop a harvesting solution: If you can’t freeze your crystals: 1. You need to develop a harvesting solution: try 2% higher concentration of precipitant than is in the drop. Slowly add a few microlitres, and watch the crystals for 2 hours. 2. If the crystals took e. g. 3 months to grow, try 10% higher conc. than is in the drop. 3. If you are unsuccessful, try including protein at the same conc. as the original experiment. 4. Loop onto a coverslip, and mount into a capillary. Microbatch seminar- slide 72 Douglas Instruments Harvesting Crystals from Microbatch

 Almost all protein crystallization experiments have at least 4 parameters: 1. Protein concentration Almost all protein crystallization experiments have at least 4 parameters: 1. Protein concentration 2. Precipitant concentration 3. p. H 4. Temperature 5. Additive ? ……………. Microbatch seminar- slide 73 Douglas Instruments Multivariate experimental design

 Microbatch seminar- slide 74 Douglas Instruments Central Composite design Microbatch seminar- slide 74 Douglas Instruments Central Composite design

 Microbatch seminar- slide 75 Douglas Instruments Box-Behnken design Microbatch seminar- slide 75 Douglas Instruments Box-Behnken design

 Microbatch seminar- slide 76 Douglas Instruments The autodesign function of XSTEP …. Microbatch seminar- slide 76 Douglas Instruments The autodesign function of XSTEP ….

 Microbatch seminar- slide 77 Douglas Instruments …. automatically fills a “spreadsheet” … Microbatch seminar- slide 77 Douglas Instruments …. automatically fills a “spreadsheet” …

Microbatch seminar- slide 78 Douglas Instruments …. and XSTEP executes it. Microbatch seminar- slide 78 Douglas Instruments …. and XSTEP executes it.

Microbatch seminar- slide 79 Yaakov showed me … Douglas Instruments And finally - Microbatch seminar- slide 79 Yaakov showed me … Douglas Instruments And finally -

Microbatch seminar- slide 80 Douglas Instruments What Yaakov saw Microbatch seminar- slide 80 Douglas Instruments What Yaakov saw

Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Vaughan Mills, Richard Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Vaughan Mills, Richard Briggs Microbatch seminar- slide 81 Douglas Instruments Imperial College, London: Professor David M. Blow, Patrick Shaw Stewart, Dennis Maeder, Naomi Chayen

 Microbatch seminar- slide 82 Douglas Instruments How can we do vapor diffusion as Microbatch seminar- slide 82 Douglas Instruments How can we do vapor diffusion as easily as microbatch?

 Number of crystals 0. 5 M AS 1. 0 M AS Days Microbatch Number of crystals 0. 5 M AS 1. 0 M AS Days Microbatch seminar- slide 83 Douglas Instruments Vapor Diffusion effect demonstrated by increasing reservoir concentration

Step 1. “Primary Screen. ” Approx. 30 -dimensional search. E. g. Sparse Matrix or 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 84 Douglas Instruments Experimental Design Steps