Dear Enrico, You are right that the trick has its limitations and I am aware of it. However, it might not be as bad as you think. Fiddling with the crystallization buffer or transferring crystals to different buffers also causes stress to the crystals and in many cases loss of resolution. If it turns out that the reservoir solution freezes ok (or the crystallization drop itself if that is feasible), I would risk, as I said, trying to freeze a crystal directly from the drop without any further manipulations. Why try to cryoprotect a crystal when it is not necessary? If that does not work, I would go for more elaborate protocols, which, as far as I know, also do not have 100% guarantee of success. In that case I would also consult the literature like the excellent paper you mention.
Best, Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Enrico Stura Gesendet: Dienstag, 27. August 2013 11:58 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] cryoprotection Herman, The trick you suggest is not as valid as you may think. The ice rings can originate from the crystal itself. If you crystallize in a high concentration PEG precipitant you will avoid ice rings, but if you transfer or soak your crystals in the same solution the high molecular weight PEG will not enter the crystal lattice and you will still get ice rings. I have a picture of this in: Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth & Design, e-print http://pubs.acs.org/doi/full/10.1021/cg301531f PDF: Figure3 Page G. So cryoprotectants need to penetrate the crystal lattice to prevent ice rings, but even in the presence of ice rings the data can be used. Regarding optimization: The main problem you encounter in cryoprotection is that some compounds like glycerol and ethylene glycol solubilize protein crystals, but if you create a mixture of various compounds that is precipitation-solubilization neutral, then there is no real need for optimization. Enrico. On Tue, 27 Aug 2013 08:30:28 +0200, <herman.schreu...@sanofi.com> wrote: > A trick I like is just to freeze the reservoir solution or would-be > cryo-solution without a crystal present. If the frozen solution stays > clear and does not show ice rings on e.g. a home source, it is worth > trying. Otherwise, the solution needs optimization. > Herman > > > -----Ursprüngliche Nachricht----- > Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von > Uday Kumar > Gesendet: Freitag, 23. August 2013 19:52 > An: CCP4BB@JISCMAIL.AC.UK > Betreff: [ccp4bb] cryoprotection > > Hello > > Can anyone suggest a cryoprotectant for the following crystallization > condition > > 0.2-0.4M sodium formate > > ~20% PEG 3350 > > 0-25 mM Nickel > > 0-100 mM Malonate > > Thank you > > with regards > uday -- Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=en&user=Kvm06WIoPAsC&pagesize=100&sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
