Dear all, Thank you to all those who responded to my question about freezing crystals grown in isopropanol. To recap: - my crystals grew in 25% isopropanol, plus some citrate buffer at pH4.5 (no other ingredients). - my crystals grew at room temperature, in 1micro-litre sitting drops, in Innovaplate SD-2 crystallization plates. Summary of comments/suggestions from CCP4BB: - harvesting crystals in the cold room to reduce isopropanol evaporation - use the mother liquor (i.e. isopropanol) as cryoprotectant - overlay the drop with oil to reduce isopropanol evaporation; some oils can also serve as a cryoprotectants - bathe the work area in a stream of air or nitrogen that has been pre-saturated with the reservoir solution - using special crystallization plates, such as Vapor Batch plates - Trying to grow the same crystals using a different precipitant (e.g. butanediol, PEG6000) Here is what I did: - working in the cold room helped a little, but there was still significant isopropanol evaporation - covering the drop with oil reduced isopropanol evaporation and made harvesting the crystals relatively straightforward - I tried the following cryoprotectants * Freezing crystals directly from mother liquor: massive ice rings, very poor diffraction * 20-30% glycerol: substantial ice rings * 20-30% ethylene glycol: substantial ice rings * Perfluoropolyether oil: a reasonable cryoprotectant, minimal ice rings * 20% MPD: minimal ice rings Using the MPD condition, we were able to collect a dataset to 2.8A resolution.
Thanks again for all your advice -- best wishes, Chris
