douglas instruments microbatch seminar- slide 1 should we be doing more crystallization by the...

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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. Phase diagrams

3. Comparisons of microbatch and vapor diffusion

4. Case studies

5. Experimental design

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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

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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

4. Cheap!

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Microbatch seminar- slide 12

Microbatch crystallization

Volume of well - 12 microlitres

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Microbatch seminar- slide 13

Microbatch crystallization

Volume of drop - 0.2 to 2 microlitres

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Microbatch seminar- slide 14

Microbatch crystallization

(2-bore)microtip

Oil

Sample

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Microbatch seminar- slide 15

Microbatch crystallization

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Microbatch seminar- slide 16

Microbatch crystallization

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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

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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

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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

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Microbatch seminar- slide 20

Phase diagram of a protein

[Protein]

[Precipitant]

clear

precipitate

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Microbatch seminar- slide 21

Phase diagram of a protein

[Protein]

[Precipitant]

clear

precipitate

nucleation

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Microbatch seminar- slide 22

Phase diagram of a protein

[Protein]

[Precipitant]

clear

precipitate

nucleation

metastable zone

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Microbatch seminar- slide 23

Phase diagram of a protein

[Protein]

[Precipitant]

c

p

n

m.z. Vapor diffusion

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Microbatch seminar- slide 24

Phase diagram of a protein

[Protein]

[Precipitant]

c

p

n

m.z. v.d.

Microbatch

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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.)

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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

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Microbatch seminar- slide 27

What % of protein should you use?

[Protein]

[Precipitant]

n

m.z.

Microbatch with Si. / Par.:

Precipitant saturated

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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%

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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%

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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

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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

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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.

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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

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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.

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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.

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Microbatch seminar- slide 36

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 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

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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

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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

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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

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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

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Microbatch seminar- slide 42

[Protein]

[PEG 4K]

Reservoir – 16.5 %Droplet – 15.5 %

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Microbatch seminar- slide 43

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

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Microbatch seminar- slide 44

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

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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.

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Microbatch seminar- slide 46

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

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Microbatch seminar- slide 47

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

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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

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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 ? …………….

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Microbatch seminar- slide 50

Central Composite design

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Microbatch seminar- slide 51

Box-Behnken design

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Microbatch seminar- slide 52

The autodesign function of XSTEP ….

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Microbatch seminar- slide 53

…. automatically fills a “spreadsheet” …

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Microbatch seminar- slide 54

…. and XSTEP executes it.

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Microbatch seminar- slide 55

ORYX (arabian)

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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.