introduction to microbatch protein crystallization patrick shaw stewart imperial college, london:

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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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What is microbatch crystallization?

• 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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ORYX 6 crystallization system

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Liquid-handling channel

Motorized Hamilton gas-tight syringe (water) X 5

to microtip

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ORYX 6 crystallization system

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Large-volume tip for oil

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Left-hand tip:

2-bore Microtip – screening

5-bore Microtip – optimization

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End of a 5-bore microtip

0.15 mm

0.9 mm

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Microbatch screening

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2-bore Microtip – screening

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Microbatch screening – dispensing cycle

Screening solutionsScreening solutions

Target Target plateplate

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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 screening – “sip and spit”

(1)(1)1. Pick up 100 nl screening solution

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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 optimization

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5-bore Microtip – optimization

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Microbatch optimization – dispensing cycle

(1)(1)1. Dispense five

solutions together

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Microbatch optimization – dispensing cycle

(2) oil(2) oil1. Dispense five solutions together

2. Oil

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Central Composite design

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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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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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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 20 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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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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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]

[Precipitant]

p

m.z.

1. Determination of phase diagram

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A few good quality crystals were obtained

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[Protein]

[PEG 4K]

Edge of nucleation – 16 % PEG

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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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[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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Exactly the same conditions – but with no seeding solution - gave poor crystals

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

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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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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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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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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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Crystals grown by VB with isopropanolCrystals grown by VB with isopropanol

1918 H1 catalase

Low ionic strength PEG screen, Sigma

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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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Harvesting Crystals from Microbatch

James Liu - University of Georgia

Jeroen Mesters - University of Luebeck

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James Liu – B.C. Wang’s group, University of Georgia

High-throughput crystallization for structural genomics

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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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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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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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Oryx at the Biblical Zoo in Jerusalem

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ORYX (arabian)

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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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And finally - Yaakov showed me …

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What Yaakov saw

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How can we do vapor diffusion as easily as microbatch?

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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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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.

introduction to microbatch protein crystallization patrick shaw stewart imperial college, london:

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