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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 What is 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 5 Douglas Instruments Microbatch crystallization
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 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
• Versatile – optimization, additives etc. Microbatch seminar- slide 9 Douglas Instruments Oryx Crystallization System
• 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 Douglas Instruments Oryx Crystallization System
• 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 – 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 Douglas Instruments How does Oryx work?
Microbatch seminar- slide 15 Douglas Instruments Sitting Drop
• 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 Microbatch seminar- slide 17 Douglas Instruments Sitting Drop - preparation
• 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 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 – dispensing cycle
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) 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 24 Douglas Instruments IMPAX 1 -5 crystallization system
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 27 Douglas Instruments ORYX 6 crystallization system
Microbatch seminar- slide 28 Douglas Instruments ORYX 6 crystallization system
Microbatch seminar- slide 29 Douglas Instruments Microbatch screening
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 – dispensing cycle
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 Microbatch seminar- slide 33 (1) Douglas Instruments Microbatch screening – dispensing cycle
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
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 Douglas Instruments Microbatch optimization – dispensing cycle
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 Douglas Instruments Microbatch optimization – dispensing cycle
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 of a protein
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. Douglas Instruments Phase diagram of a protein
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. (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. (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 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% [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 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 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 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 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 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 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. 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 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 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 of phase diagram
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 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 Instruments Droplet – 15. 5 %
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 no seeding solution - gave poor crystals
(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 grown at 18ºC
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 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 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 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 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 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: 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 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 75 Douglas Instruments Box-Behnken design
Microbatch seminar- slide 76 Douglas Instruments The autodesign function of XSTEP ….
Microbatch seminar- slide 77 Douglas Instruments …. automatically fills a “spreadsheet” …
Microbatch seminar- slide 78 Douglas Instruments …. and XSTEP executes it.
Microbatch seminar- slide 79 Yaakov showed me … Douglas Instruments And finally -
Microbatch seminar- slide 80 Douglas Instruments What Yaakov saw
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 easily as 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 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
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