Vapor batch crystallization using volatile organic solvents

1. Vapor batch crystallization using 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

2. Crystallisation of NTD ofN-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.

3. Attempts 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.

4. Vapor 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.)

5. Vapor Batch trays (Douglas Instruments)

6. Procedure • Droplets (2l) 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 18C. • Next day, the 10% isopropanol solution was replaced by 20% isopropanol

7. This method was used to grow crystals 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

8. NTD 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.

9. Effect of isopropanol as an additive • Low ionic strength PEG screens • Vary pH and PEG concentration • +/- isopropanol or other volatile organic in the moat. • High salt screens • Use 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.

10. Crystals grown by VB with isopropanol catalase Haemagglutinin LowionicstrengthPEGscreen

11. Advantages of vapor batch cf. vapor diffusion • Improved crystal stability • Easier crystal handling • Better diffraction from crystals grown under paraffin/silicone oil mixture.