douglas instruments microbatch seminar- slide 1 introduction to microbatch protein crystallization...

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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

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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.