Microbatch crystallisation: worth a try?? Allan D’Arcy, Aengus Mac Sweeney

Microbatch Crystallisation  Batch method: where the conditions to produce crystals are pre-determined, no concentration of the solution is required.  Microbatch: using small volumes of protein and precipitating agent under a layer of paraffin oil (Chayen et. al. 1990)  Automated microbatch: Douglas Instruments develops a robot for setting up crystallisation drops under paraffin oil.(1992)  Modified Microbatch or microbatch diffusion: By modifying the oils used a system is established which allows concentration of the drop. (D’Arcy et.al.1996)

The methods Protein molecules in solution Increase protein concentration and add precipitating agent, molecules may self associate Reservoir e.g. 50% ammonium sulphate Protein + precipitating agent Vapour diffusion hanging drop

Mimic vapour diffusion under oil

Automation: vapour diffusion

Automation: Microbatch Twin tube for prot+ ppt.agent Screen solutions 72 well plate

Comparison of modified microbatch and vapour diffusion (11 different proteins)  Vapour diffusion  62 hits  11% success  Number of crystals with 3 dimension (thick plates, rods or prisms) =32  Mod.Microbatch  104 hits  19.6% success  Number of crystals with 3 dimension (thick plates, rods or prisms) = 58

Tight sealing cover slip Reservoir drop  l 6  l protein +reservoir drop Going from modified microbatch to vapour diffusion Occasionally it is difficult to reproduce crystallisation from microbatch in vapour diffusion even if identical solutions are used. One possibility is to use a low volume reservoir vapour diffusion in a sitting drop plate, based on the method described by George De Titta (RAMC 1997) If this fails add DTT!!!!

Al’s oil no reservoir drops dry out ~2 months Silicone oil no reservoir drops dry out Paraffin oil no reservoir drops are stable, different crystal morphology Modified microbatch drawbacks After 2 months

Al’s oil + reservoir Silicone oil + reservoir Paraffin oil + reservoir Possible Solution: enclose with a reservoir or add paraffin oil After 2 months

Some crystals grown in modified microbatch (non lysozyme)

Small volumes are possible 100nl total 200nl total 400nl total 100nl drop of aldolase with crystals at low and high magnification

Small volumes-mega screens?  George De Titta and Joe Luft have developed a mother daughter technology for high throughput screening using the microbatch method.  1536 well plates are used meaning that in principle 40,000 experiments could be set up in a 24hr period.  Next problem is who scores the plates!!!

Microbatch makes automation easy  No greasing is required  Drops are not exposed to air for any long periods of time  Small volumes are possible with simple liquid handling devices  No reservoir solutions need to be pipetted  Solutions of different viscosity can be handled easily.

Higher supersaturation may result in more hits  Modified microbatch has no end point if no reservoir is included or extra oil added, drops will eventually dry out.  This means however that higher concentrations of both precipitating agent and protein can be reached compared to the vapour diffusion experiment  Protein needs to be stable over these long periods

Microbatch helps to prevent oxidation Permanganate exposed to air changes colour Microbatch drop 24hrs Vapour diffusion drop 24hrs

Conclusions Modified microbatch is a useful alternative to standard vapour diffusion for screening. It can work with small volumes of protein It can be scaled up to 1536 well plates It can be made high throughput at low cost It is suitable for temperature gradient experiments Offers some protection against protein oxidation