douglas instruments microbatch seminar- slide 1 should we be doing more crystallization by the...
TRANSCRIPT
Dou
gla
s
Instr
um
en
ts
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 2
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 3
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 4
What is the microbatch method?
• Crystallization in small drops under oil
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 5
What is the microbatch method?
• Crystallization in small drops under oil
• 100 + 100 nl to 1+1 µl
Dou
gla
s
Instr
um
en
ts
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 7
Why is microbatch a good idea?
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 8
Why is microbatch a good idea?
1. Easy
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 9
Why is microbatch a good idea?
1. Easy
2. Gives better crystals in many cases – especially in screening
Dou
gla
s
Instr
um
en
ts
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
Dou
gla
s
Instr
um
en
ts
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
4. Cheap!
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 12
Microbatch crystallization
Volume of well - 12 microlitres
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 13
Microbatch crystallization
Volume of drop - 0.2 to 2 microlitres
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 14
Microbatch crystallization
(2-bore)microtip
Oil
Sample
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 15
Microbatch crystallization
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 16
Microbatch crystallization
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 17
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 18
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 19
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 20
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 21
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 22
Phase diagram of a protein
[Protein]
[Precipitant]
clear
precipitate
nucleation
metastable zone
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 23
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. Vapor diffusion
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 24
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. v.d.
Microbatch
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 25
Phase diagram of a protein
[Protein]
[Precipitant]
c
p
n
m.z. v.d..
M.B.(paraffin)
M.B.(par./si.)
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 26
Phase diagram of a protein
[Protein]
[Precipitant]
p
n
m.z. v.d.
M.B.(paraffin)
OPTIMIZATION
M.B.(par./si.)
SCREENING
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 27
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Precipitant saturated
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 28
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 29
What % of protein should you use?
[Protein]
[Precipitant]
n
m.z.
Microbatch with Si. / Par.:
Protein stock
Precipitant stock
Precipitant saturated
50%66%
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 30
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 31
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 32
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.
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 33
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 34
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.
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 35
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.
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 36
OPTIMIZATION: about 50:50
• In microbatch, there tends to be more precipitation initially; this may result in more nucleation
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 37
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 38
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 39
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 40
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 41
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
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 42
[Protein]
[PEG 4K]
Reservoir – 16.5 %Droplet – 15.5 %
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 43
Reproducible good quality crystals wereobtained with microseeding. Crystals diffracted to 2Å
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 44
Vapor Batch trays Vapor Batch trays (Douglas Instruments)(Douglas Instruments)
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 45
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.
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 46
Crystals obtained at 4ºC(Lesley Haire, Imperial College)
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 47
Crystals nucleated for 1 hr 4ºC, then grown at 18ºC
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 48
1. What is the microbatch method?
2. Phase diagrams
3. Comparisons of microbatch and vapor diffusion
4. Case studies
5. Experimental design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 49
Multivariate experimental design
Almost all protein crystallization experiments have at least 4 parameters:
1. Protein concentration
2. Precipitant concentration
3. pH
4. Temperature
5. Additive ? …………….
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 50
Central Composite design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 51
Box-Behnken design
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 52
The autodesign function of XSTEP ….
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 53
…. automatically fills a “spreadsheet” …
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 54
…. and XSTEP executes it.
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 55
ORYX (arabian)
Dou
gla
s
Instr
um
en
ts
Microbatch seminar- slide 56
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.