Re: [ccp4bb] Cryo-protectant

[Roger Rowlett]

I usually try the following, in order: 1. Transfer crystals to mother liquor + 30% glycerol or ethylene glycol (sometimes lower depending on crystallization solution). This did not work for you. 2. Transfer crystals to mother liquor + 30% glucose (or try sequential soaks in M.L. + 15% and then 30% glucose. Just a few sec/min is usually enough). Glucose or other sugars often work. when glycerol or EG fails. 3. Try the "no-fail" in situ cryo method, which is a gradual buildup of cryoprotectant. See http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo . This is very gentle, and often works when #1 and #2 does not, but in our hands nearly always increases mosaicity. (But mosaic is better than no diffraction.) 4. Try dragging crystals though paratone-N to remove surface water from the crystal. This actually nearly always works for us, but is more fussy than #1 or #2, and it is easier to damage crystals during manipulation because of the viscosity of the oil. I normally plunge protected crystals into LN2 after mounting. Ice rings are a good indication of poor cryoprotection, but lack of diffraction could just be your crystals, too. For our latest dataset, we just sorted through 38 crystals until we found a good one. The key, as it turned out, is that all of our beautiful large crystals were apparently difficult to visualize disordered stacks of plates (we didn't notice this until some fractured during cryo-soaks) whereas some of the small crystals were actually single crystals. We selected a decently diffracting small one and took loooooooooooong frames to get a good data set. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Liew Chong Wai wrote: Hi all,   Good day I used MPD as a cryoprotectant (20%, 30%) for my crystal. However, there is no diffraction signal at all. Without the MPD cryo, i still manage to get 5angstrom, but it has very strong ice-ring signal. I used glycerol (15%, 20%, 25% and 30%) before, but it cracked the crystal.   Please advice.   Thanks    Liew