Hi Fengxiale, Have you tried no cryoprotection? 10% PEG4K is probably too low but you may get lucky. Since your crystal is already in PEG, you may try increasing the concentration of the PEG or try just plain ethylene glycol, which may be more gentle on your crystal than sticking it in glycerol. You say you started with 25% glycerol and slowly decreased to 12.5%. Are you doing this by dialysis or by just sequentially transferring your crystal from one solution to the next? Dialysis is much more gentle and you should be going the other direction - i.e. from low conc. of cryoprotectant to high conc (not high to low like you did). How long are you soaking your crystal in the cryoprotectant solution? Do crystals crack immediately or does it take some time? Oftentimes just a quick dip is sufficient. How well does your crystal diffract at room temperature? Perhaps 3.2 A is the limit even in the absence of freezing.
Good luck, Eric __________________________ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Tue, 1 Sep 2009, Fengxia Liu wrote:
Dear All, Could you please help me solve this problem? I have a sensitive crystal, the mother liquor is 10% PEG4k+ 100 mM Tris-buffer pH 8.5, crystal is big and good but very sensitive, when i put it in cryoprotectants, cracking happened. First i used artificial mother liquor + 25% v/v glycerol, slowly decrease to + 12.5% v/v glycerol, all crystals cracked after immersed, finally i tried 50% mineral oil + 50% paratone, it still cracked (4- 5 cracks, but not broken) . even this cracked crystal can give me 3.2 angstrom diffraction, so no cracked crystal might give me 2.0+ angstrom diffraction. Now i don't have many crystals to try so many cryoprotectants, so anybody has experience on this? any suggestion would be greatly appreciated. Thank you. Fengxiale
