Further to the other contributions to this discussion, please note that the Oxford Diffraction PX Scanner system - for diffraction quality assessment of crystals in situ in the crystallisation plate, can also play a powerful role in all of this. Thus, in addition to differentiating salt from protein crystals and non-invasively identifying the 'best diffracting' crystal in a plate, by in situ diffraction examination of a crystal before and after the addition of cryo-protectant to the droplet, then any problems actually created by the cryo- can be identified.
Thanks. Marcus Winter (Oxford Diffraction) -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Andy Torelli Sent: 24 April 2009 16:46 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cryo-protectant Hi Liew, There have already been some very good suggestions. I agree with Tim Gruene that a great starting point is to test the diffraction properties of your crystal at room temperature. This can serve as a baseline for comparing/evaluating cryoprotecting agents and methods. You can also test your potential cryoprotection solutions to see if they freeze clear or even take a few X-ray snapshots of them to confirm there are no ice rings. There are lots of publications that can be helpful. One very helpful reference is: Garman, E.F. and Doublie, S. Cryocooling of Macromolecular Crystals: Optimisation Methods. Methods in Enzymology (2003) 368, 188-216. Be aware that, as mentioned previously, the method for freezing can make a difference (e.g. freezing in cold-stream vs. plunging in nitrogen). An obvious difference is different cooling rates (you can find references for this) or less obvious reasons, for example differences in dehydration that occur during longer/shorter transfer through air from drop to cold-source for either method. Finally, you can check out the database for cryoprotecting solutions: http://idb.exst.jaxa.jp/db_data/protein/search-e.php Good luck, -Andy -- ============================================= Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University ============================================= On 4/24/2009 10:44 AM, Liew Chong Wai wrote: > Hi all > > Thanks for your precious suggestions and ideas. > The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M > MgCl.6H2O, 35% PEG3350 > Now, my crystal seem ok in 20% ethylene glycol, but only after a couple > minutes of dehydration at room temperature. For sure, i will try other > cryoprotectant that was suggested here. > I just wondering why MPD kills the crystal. > Many thanks > > *LIEW* > > > > > > > -- ============================================= Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University =============================================
