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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1. What is the microbatch method?

2. Phase diagrams

3. Comparisons of microbatch and vapor diffusion

4. Case studies

5. Experimental design

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2. Phase diagrams


4. Case studies


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What is the microbatch method?

• Crystallization in small drops under oil

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• 100 + 100 nl to 1+1 µl

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• 100 + 100 nl to 1+1 µl

• The oil prevents evaporation

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Why is microbatch a good idea?

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1. Easy

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1. Easy

2. Gives better crystals in many cases – especially in screening

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1. Easy


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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1. Easy


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 crystallization

Volume of well - 12 microlitres

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Volume of drop - 0.2 to 2 microlitres

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(2-bore)microtip

Oil

Sample

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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


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


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 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


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


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


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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2. Phase diagrams


4. Case studies


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Phase diagram of a protein

[Protein]

[Precipitant]

clear

precipitate

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[Protein]

[Precipitant]

clear

precipitate

nucleation

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[Protein]

[Precipitant]

clear

precipitate

nucleation

metastable zone

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[Protein]

[Precipitant]

c

p

n

m.z. Vapor diffusion

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[Protein]

[Precipitant]

c

p

n

m.z. v.d.

Microbatch

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[Protein]

[Precipitant]

c

p

n

m.z. v.d..

M.B.(paraffin)

M.B.(par./si.)

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[Protein]

[Precipitant]

p

n

m.z. v.d.

M.B.(paraffin)

OPTIMIZATION

M.B.(par./si.)

SCREENING

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What % of protein should you use?

[Protein]

[Precipitant]

n

m.z.

Microbatch with Si. / Par.:

Precipitant saturated

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[Protein]

[Precipitant]

n

m.z.


Protein stock

Precipitant stock


50%

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[Protein]

[Precipitant]

n

m.z.


Protein stock

Precipitant stock


50%66%

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2. Phase diagrams


4. Case studies


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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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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%


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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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



N. Noordeen and S. Cowan-Jacob. Novartis Pharma AG. http://www.hamptonresearch.com/stuff/ppt_files/P6.ppt

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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%




Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

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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



Misuaki Sugahara, Riken Harima Institute, SPring8. Personal communication.

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OPTIMIZATION: about 50:50

• In microbatch, there tends to be more precipitation initially; this may result in more nucleation

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• 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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• 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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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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2. Phase diagrams


4. Case studies


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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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[Protein]

[PEG 4K]

Reservoir – 16.5 %Droplet – 15.5 %

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Reproducible good quality crystals wereobtained with microseeding. Crystals diffracted to 2Å

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Vapor Batch trays Vapor Batch trays (Douglas Instruments)(Douglas Instruments)

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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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Crystals obtained at 4ºC(Lesley Haire, Imperial College)

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Crystals nucleated for 1 hr 4ºC, then grown at 18ºC

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2. Phase diagrams


4. Case studies


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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 ? …………….

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Central Composite design

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Box-Behnken design

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The autodesign function of XSTEP ….

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…. automatically fills a “spreadsheet” …

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…. and XSTEP executes it.

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ORYX (arabian)

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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.

Douglas Instruments Microbatch seminar- slide 1 Should we be doing more crystallization by the microbatch method? Patrick Shaw Stewart Imperial College,

Documents

microbatch slide

oil slide

screening slide

easy slide

microbatch method

microlitres slide

evaporation slide

comparisons of microbatch