Re: [ccp4bb] cryo protection

[Roger Rowlett]

Len, We have run into this problem from time to time, and it is very frustrating. Here are some things to try, some of which you may have done already: Grow crystals in a small percentage of the cryoprotectant (e.g., 5-10% glycerol). This often allows crystal transfer into a cryo drop without cracking. (Almost never works for us, though.) Do your crystal transfers in the cold room. This slows evaporation markedly, and may prevent crystal cracking. (This works for us some of the time.) Transfer your crystals to gradually higher cryoprotectant concentrations (e.g., to 15% glycerol, then 30% glycerol). (Fiddly, and the crystals get handled a lot, but often works.) Use different cryoprotectants. We almost always have fewer cracking issues with glucose than glycerol, but YMMV. Avoid transferring the crystal from the drop at all. Just add cryoprotectant to the drop. Even better, add cryoprotectant to the drop gradually, while keeping the drop humidified over well solution. This is our "No-fail" method (this is usually, but not always successful): http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals#No_fail_cryoprotection We typically use glucose in this method, but in principle you could try glycerol, MPD, PEG-400, or sodium formate, etc. Otherwise, you can try to grow out of a cryo condition that doesn't need extra cryoprotectant (been there done that) or give up and shoot at room temp in-house. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor 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...@colgate.edu On 10/26/2011 12:46 PM, Leonard Thomas wrote: Hi All, I have run into a very sensitive crystals system when it comes to cryo protecting them.  I have run through the usual suspects and trays are going to be setup with a cryo protectant as part of crystallization cocktail.  The one problem that  seems to be occurring is that the crystals crack as soon as they are transfered out of the original drop.  I am running out of ideas and really would love some new ones. Thanks in advance. Len Leonard Thomas Ph.D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry Stephenson Life Sciences Research Center 101 Stephenson Parkway Norman, OK 73019-5251 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: (405)325-1126 Lab: (405)325-7571