introduction to microbatch protein crystallization patrick shaw stewart imperial college, london:
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Introduction to microbatch protein crystallization 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. - PowerPoint PPT PresentationTRANSCRIPT
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Microbatch seminar- slide 1
Introduction to microbatch protein crystallization
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
What is microbatch crystallization?
• Crystallization in small drops under oil
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Microbatch seminar- slide 3
What is microbatch crystallization?
• Crystallization in small drops under oil
• 100 + 100 nl to 1+1 µl
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Microbatch seminar- slide 4
What is microbatch crystallization?
• Crystallization in small drops under oil
• 100 + 100 nl to 1+1 µl
• The oil prevents evaporation
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Microbatch seminar- slide 5
Why is microbatch a good idea?
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Microbatch seminar- slide 6
Why is microbatch a good idea?
1. Easy
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Microbatch seminar- slide 7
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 8
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 9
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!
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Microbatch seminar- slide 10
Microbatch crystallization
Volume of well - 12 microlitres
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Microbatch seminar- slide 11
Microbatch crystallization
Volume of drop - 0.2 to 2 microlitres
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Microbatch seminar- slide 12
Microbatch crystallization
(2-bore)microtip
Oil
Sample
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Microbatch seminar- slide 13
Microbatch crystallization
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Microbatch seminar- slide 14
Microbatch crystallization
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Microbatch seminar- slide 15
Microbatch optimization – print outRow 1
50mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.063M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 095% 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 0
Row 250mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
Row 350mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
Row 450mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
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Microbatch seminar- slide 16
Microbatch optimization – print outRow 1
50mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.063M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35
100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 095% 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 0
Row 250mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
Row 350mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
Row 450mg/ml BSA 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06 1.06
3M NaAc pH7 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35100% Pure green dye 0 0 0 0 0 0 0 0 0 0 0 0
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 0
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Microbatch seminar- slide 17
ORYX 6 crystallization system
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Microbatch seminar- slide 18
Liquid-handling channel
Motorized Hamilton gas-tight syringe (water) X 5
to microtip
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Microbatch seminar- slide 19
ORYX 6 crystallization system
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Microbatch seminar- slide 20
Large-volume tip for oil
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Microbatch seminar- slide 21
Left-hand tip:
2-bore Microtip – screening
5-bore Microtip – optimization
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Microbatch seminar- slide 22
End of a 5-bore microtip
0.15 mm
0.9 mm
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Microbatch seminar- slide 23
Microbatch screening
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Microbatch seminar- slide 24
2-bore Microtip – screening
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Microbatch seminar- slide 25
Microbatch screening – dispensing cycle
Screening solutionsScreening solutions
Target Target plateplate
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Microbatch seminar- slide 26
Sitting Drop - preparation
Air bubble
Protein slug
• Suck up protein required for experiment + 0.25 µl
• Suck air bubble in second bore – for transfer
Air bubble
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Microbatch seminar- slide 27
Microbatch screening – “sip and spit”
(1)(1)1. Pick up 100 nl screening solution
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Microbatch seminar- slide 28
Microbatch screening – “sip and spit”
(2)(2) (1)(1)1. Pick up 100 nl screening solution
2. Transfer to microbatch drop and add protein
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Microbatch seminar- slide 29
Microbatch optimization
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Microbatch seminar- slide 30
5-bore Microtip – optimization
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Microbatch seminar- slide 31
Microbatch optimization – dispensing cycle
(1)(1)1. Dispense five
solutions together
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Microbatch seminar- slide 32
Microbatch optimization – dispensing cycle
(2) oil(2) oil1. Dispense five solutions together
2. Oil
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Microbatch seminar- slide 33
Central Composite design
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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
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
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Microbatch seminar- slide 36
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
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Microbatch seminar- slide 37
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
metastable zone
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Microbatch seminar- slide 38
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. Vapor diffusion
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Microbatch seminar- slide 39
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. v.d.
Microbatch
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Microbatch seminar- slide 40
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 41
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 42
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Precipitant saturated
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Microbatch seminar- slide 43
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 44
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 45
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 46
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 47
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 48
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 49
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 50
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 51
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 52
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 20 structures solved in the last few years, 5 relied on microbatch
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Microbatch seminar- slide 53
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 54
Crystals obtained at 4ºC(Lesley Haire, Imperial College)
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Microbatch seminar- slide 55
Crystals nucleated for 1 hr 4ºC, then grown at 18ºC
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Microbatch seminar- slide 56
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 57
[Protein]
[Precipitant]
p
m.z.
1. Determination of phase diagram
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Microbatch seminar- slide 58
A few good quality crystals were obtained
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Microbatch seminar- slide 59
[Protein]
[PEG 4K]
Edge of nucleation – 16 % PEG
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Microbatch seminar- slide 60
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 61
[Protein]
[PEG 4K]
Reservoir – 16.5 %Droplet – 15.5 %
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Microbatch seminar- slide 62
Reproducible good quality crystals wereobtained with microseeding. Crystals diffracted to 2Å
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Microbatch seminar- slide 63
Exactly the same conditions – but with no seeding solution - gave poor crystals
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Microbatch seminar- slide 64
Vapor batch crystallization Vapor batch crystallization using volatile organic solventsusing volatile organic solvents
Lesley Haire
Division of Protein Structure,
National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK
Winner of the competition for the Best Use of the Douglas Vapor Batch Plate
First round - January 2005
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Microbatch seminar- slide 65
Crystallisation of NTD ofCrystallisation of NTD ofN-MLV capsidN-MLV capsid
Crystals were grown from hanging drops - Hampton Crystal Screen no.40, 20% PEG 4000, 20% v/v isopropanol, 0.1M Nacitrate pH 5.6 20mg/ml protein in the drops
Major problem - harvesting the crystals in the presence of isopropanol.
The crystals disintegrated as soon as the coverslip was opened.
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Microbatch seminar- slide 66
Attempts to overcome the problemAttempts to overcome the problem
Using sitting drops,
oil over the drops,
and handling crystals using constant humidity were only partially successful.
In microbatch experiments under oil, crystals were not stable and dissolved after a couple of days.
Crystals that were X-rayed had high mosaicity and could not be used for structure solution.
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Microbatch seminar- slide 67
Vapor Batch trays Vapor Batch trays (Douglas Instruments)(Douglas Instruments)
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Microbatch seminar- slide 68
ProcedureProcedure Droplets (2l) dispensed under a mixture of silicone/
paraffin oil using IMPAX 1-5 crystallisation robot.
A 6x4 spreadsheet was set up with XSTEP software varying; protein, 16-22 mg/ml; PEG 3350, 13-16%; all wells had 0.1M sodium citrate pH 5.6.
10% isopropanol was pipetted into the tray’s “moat” and the drops equilibrated overnight at 18C.
Next day, the 10% isopropanol solution was replaced by 20% isopropanol
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Microbatch seminar- slide 69
This method was used to grow crystals This method was used to grow crystals of NTD N-MLV capsid protein:of NTD N-MLV capsid protein:
Crystals appeared after a couple of days.
Typically they were harvested and frozen after 10 days.
Crystals were very stable in drops for at least 6 months.
Diffraction to 1.9Å with low mosaicity.
Crystals did not grow in the controls without isopropanol in the moat.
Capsid protein was provided by Nehar Mortuza
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Microbatch seminar- slide 70
NTD N-tropic MLV- capsid proteinNTD 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.
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Microbatch seminar- slide 71
Using “vapor batch” in screeningUsing “vapor batch” in screening
Low ionic strength PEG screensVary pH and PEG concentration +/- isopropanol or other volatile organic in the moat.
High salt screensUse AmS04 or P04, set up duplicate trays, +/-10% isopropanol in the moat.
The same principle could be used to test isopropanol or any other volatile additive with a selected screen dispensed in VB trays.
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Microbatch seminar- slide 72
Crystals grown by VB with isopropanolCrystals grown by VB with isopropanol
1918 H1 catalase
Low ionic strength PEG screen, Sigma
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Microbatch seminar- slide 73
Advantages of “vapor batch” cf. Advantages of “vapor batch” cf. vapor diffusion:vapor diffusion:
Improved crystal stability
Easier crystal handling
Better diffraction from crystals grown under paraffin/silicone oil mixture.
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Microbatch seminar- slide 74
Harvesting Crystals from Microbatch
James Liu - University of Georgia
Jeroen Mesters - University of Luebeck
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Microbatch seminar- slide 75
Harvesting Crystals from Microbatch
James Liu – B.C. Wang’s group, University of Georgia
High-throughput crystallization for structural genomics
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Microbatch seminar- slide 76
Harvesting Crystals from Microbatch
James Liu
• Microbatch is easier because the oil prevents evaporation - you can work slowly!
• You can loop straight out of the droplet through the oil.
• James’ record – he mounted 98 crystals in one day!
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Microbatch seminar- slide 77
Harvesting Crystals from Microbatch
Digression:
University of Georgia is unusual: it uses sitting drop for screening and microbatch for optimization.
This reduces the solution volumes needed – and solutions can be reused many times.
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Microbatch seminar- slide 78
Harvesting Crystals from Microbatch
1. Use a loop with a bent handle.
2. Make sure the crystal fits the loop well or the oil will drag it off.
Jeroen Mesters:
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Microbatch seminar- slide 79
Oryx at the Biblical Zoo in Jerusalem
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Microbatch seminar- slide 80
ORYX (arabian)
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Microbatch seminar- slide 81
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, Vaughan Mills
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Microbatch seminar- slide 82
And finally - Yaakov showed me …
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Microbatch seminar- slide 83
What Yaakov saw
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Microbatch seminar- slide 84
How can we do vapor diffusion as easily as microbatch?
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Microbatch seminar- slide 85
Vapor Diffusion effect demonstrated by increasing reservoir concentration
Days
Number of
crystals
0.5 M AS 1.0 M AS
0
5
10
15
20
25
30
35
0 5 10 15 20 25 30 35
Mod.MB
VD
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Microbatch seminar- slide 86
Experimental Design StepsStep 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.