Hi Jobi, I also had some crystals that were highly sensitive to glycerol. In one case, I found that DMSO at 10-15% can cryo protect it, also the crystal could grow in the presence of 10% DMSO which essentially eliminated a soaking step. In another case, I grew the crystals in the presence of 5% glycerol(it won't take DMSO this time), then the crystal could survive 30% glycerol cryo soak. Also you may try using some smaller crystals as they are easier to freeze and may still diffract decently in synchrotron. Another thing to try is to crosslink the crystals with glutaraldehyde by vapor diffusion. The crystal will partially turn to a gel and will not break when soaked with cryos. In my hand this did produce tough, diffracting crystals, but didn't solve my high mosaicity problem.
Of course, RT shooting should be tested first to make sure the poor diffraction is caused by the freezing. Zhijie From: Jobichen Chacko Sent: Wednesday, April 13, 2011 5:44 AM To: [email protected] Subject: [ccp4bb] Crystal Optimization Dear All, We got crystals for a 35 KDa protein with 323aa including His tag and linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum resolution obtained till now is 5.8Å. Tried various cryo conditions like Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The crystal tends to break in the presence of glycerol. Kindly give your suggestions to improve the resolution. Thanks. Jobi
