Douglas Instruments Microbatch seminar- slide 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
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Douglas Instruments Microbatch seminar- slide 1 Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart Imperial College,
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Microbatch seminar- slide 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
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Microbatch seminar- slide 2
1. What is the microbatch method?
2. Hardware and dispensing routines
3. Phase diagrams
4. Comparisons of microbatch and vapor diffusion
5. Case studies
6. Harvesting crystals
7. Experimental design
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Microbatch seminar- slide 3
1. What is the microbatch method?
2. Hardware and dispensing routines
3. Phase diagrams
4. Comparisons of microbatch and vapor diffusion
5. Case studies
6. Harvesting crystals
7. Experimental design
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Microbatch seminar- slide 4
What is the microbatch method?
• Crystallization in small drops under oil
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Microbatch seminar- slide 5
What is the microbatch method?
• Crystallization in small drops under oil
• 100 + 100 nl to 1+1 µl
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Microbatch seminar- slide 6
What is the microbatch method?
• Crystallization in small drops under oil
• 100 + 100 nl to 1+1 µl
• The oil prevents evaporation
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Microbatch seminar- slide 7
Why is microbatch a good idea?
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Microbatch seminar- slide 8
Why is microbatch a good idea?
1. Easy
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Microbatch seminar- slide 9
Why is microbatch a good idea?
1. Easy
2. Gives better crystals in many cases – especially in screening
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Microbatch seminar- slide 10
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
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Microbatch seminar- slide 11
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
• Suck up protein required for experiment + 0.25 µl
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Microbatch seminar- slide 32
Sitting Drop – dispensing cycle
1. Rinse in reservoir
(1)(1)
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Microbatch seminar- slide 33
Sitting Drop – dispensing cycle
1. Rinse in reservoir
2. Move sideways and pick up clean solution
(2)(2)(1)(1)
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Microbatch seminar- slide 34
Sitting Drop – dispensing cycle
1. Rinse in reservoir
2. Move sideways and pick up clean solution
3. Dispense solution and protein (2)(2)
(1)(1)
(3)(3)
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Microbatch seminar- slide 35
Microbatch screening
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Microbatch seminar- slide 36
Microbatch screening – dispensing cycle
Screening solutionsScreening solutions
Target Target plateplate
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Microbatch seminar- slide 37
Microbatch screening – dispensing cycle
(1)(1)1. Pick up screening solution
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Microbatch seminar- slide 38
Microbatch screening – dispensing cycle
(2)(2) (1)(1)1. Pick up screening solution
2. Transfer to microbatch drop
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Microbatch seminar- slide 39
Microbatch screening – dispensing cycle
(2)(2) (1)(1)+ oil+ oil
1. Pick up screening solution
2. Transfer to microbatch drop; oil
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Microbatch seminar- slide 40
Microbatch screening – dispensing cycle
(2)(2) (1)(1)+ oil+ oil (3)(3)
1. Pick up screening solution
2. Transfer to microbatch drop; oil
3. Rinse
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Microbatch seminar- slide 41
Microbatch optimization
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Microbatch seminar- slide 42
Microbatch optimization – dispensing cycle
(1)(1)1. Dispense five
solutions together
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Microbatch seminar- slide 43
Microbatch optimization – dispensing cycle
(1)(1)+ oil+ oil1. Dispense five
solutions together
2. Oil
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Microbatch seminar- slide 44
Microbatch optimization – dispensing cycle
(1)(1)+ oil+ oil1. Dispense five
solutions together
2. Oil
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Microbatch seminar- slide 45
Microbatch optimization – dispensing cycle
(1)(1)+ oil+ oil
1. Dispense five solutions together
2. Oil
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Microbatch seminar- slide 46
Central Composite design
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Microbatch seminar- slide 47
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
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Microbatch seminar- slide 48
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
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Microbatch seminar- slide 49
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
metastable zone
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Microbatch seminar- slide 50
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. Vapor diffusion
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Microbatch seminar- slide 51
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. v.d.
Microbatch
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Microbatch seminar- slide 52
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. v.d..
M.B.(paraffin)
M.B.(par./si.)
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Microbatch seminar- slide 53
Phase diagram of a protein
[Protein]
[Precipitant]
p
n
m.z. v.d.
M.B.(paraffin)
OPTIMIZATION
M.B.(par./si.)
SCREENING
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Microbatch seminar- slide 54
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Precipitant saturated
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Microbatch seminar- slide 55
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%
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Microbatch seminar- slide 56
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%66%
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Microbatch seminar- slide 57
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
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Microbatch seminar- slide 58
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 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/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.
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Microbatch seminar- slide 59
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 48 104 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
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Microbatch seminar- slide 60
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 48 104 62 42 68%
2001
Noordeen et al.
Novartis Pharma 8 48 - 576 145 153 -8 -5% 95 103
Sugahara SPring8 6 288 100 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.
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Microbatch seminar- slide 61
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 48 104 62 42 68%
2001
Noordeen et al.
Novartis Pharma 8 48 - 576 145 153 -8 -5% 95 103
Sugahara SPring8 6 288 100 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.
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Microbatch seminar- slide 62
OPTIMIZATION: about 50:50
• In microbatch, there tends to be more precipitation initially; this may result in more nucleation
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Microbatch seminar- slide 63
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
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Microbatch seminar- slide 64
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
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Microbatch seminar- slide 65
OPTIMIZATION: about 50:50
From D’Arcy et al. A novel approach to crystallising proteins under oil. Journal of Crystal Growth 168 (1996) 175-180.
Vapor diffusion
Microbatch
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Microbatch seminar- slide 66
Crystals obtained at 4ºC(Lesley Haire, Imperial College)
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Microbatch seminar- slide 67
Crystals nucleated for 1 hr 4ºC, then grown at 18ºC
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Microbatch seminar- slide 68
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
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Microbatch seminar- slide 69
[Protein]
[Precipitant]
p
m.z.
1. Determination of phase diagram
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Microbatch seminar- slide 70
A few good quality crystals were obtained
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Microbatch seminar- slide 71
[Protein]
[PEG 4K]
Edge of nucleation – 16 % PEG
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Microbatch seminar- slide 72
2. Microseeding was used
1. A well-formed crystal was broken up in 15.5% PEG
2. The mixture was spun
3. A series of dilutions was set up using the supernatant (1:1000 worked best)
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Microbatch seminar- slide 73
[Protein]
[PEG 4K]
Reservoir – 16.5 %Droplet – 15.5 %
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Microbatch seminar- slide 74
Reproducible good quality crystals wereobtained with microseeding. Crystals diffracted to 2Å
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Microbatch seminar- slide 75
Exactly the same conditions – but with no seeding solution - gave poor crystals