Vapor batch crystallization Vapor batch crystallization using volatile organic using volatile organic

solventssolvents

Lesley HaireDivision 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

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.

Attempts to overcome Attempts to overcome the problemthe 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.

Vapor batch crystallizationVapor batch crystallization

The Vapor Batch tray is designed for both microbatch and vapor diffusion (sitting drop) crystallization. 

The tray has 96 wells in the centre and several reservoirs around the outside. 

– Water-filled reservoirs preserve microbatch crystals.

– Vapor diffusion experiments (where up to 96 wells are equilibrated against a single precipitant.)

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

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

This method was used to grow This method was used to grow crystals of NTD N-MLV capsid crystals of NTD N-MLV capsid protein: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

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.

Effect of isopropanol as an Effect of isopropanol as an additiveadditive

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.

Crystals grown by VB with Crystals grown by VB with isopropanolisopropanol

Haemagglutinin catalase

Low ionic strength PEG screen

Advantages of vapor batch Advantages of vapor batch cf. vapor diffusioncf. vapor diffusion

Improved crystal stability

Easier crystal handling

Better diffraction from crystals grown under paraffin/silicone oil mixture.