Hi Keith, I think there is a lot of advice out there on rational design of the cryoprotectant. One of the most useful in your case is one that I think originates with Elspeth Garman (but she will correct me if I'm wrong). This is osmolarity matching. I don't have a reference to hand but you can look up the osmotic pressure of the mother liquor and cryoprotectant agents (e.g. CRC Handbook of Chemistry and Physics) then modify the concentration of the mother liquor in the cryoprotectant solution to minimize the change in pressure. I think there is a chapter in Evolving Methods for Macromolecular Crystallography, NATO Science Series-II, that covers this and without the reference in front of me I think there is also a nice bit on it in methods in Molecular Biology, Garman and Owen, 2007, 364, 1-18. Again, others will correct me if I'm wrong or provide better pointers.
Approx 40% PEG 4000 should work as a cryo, see Kempkes et al., Acta Cryst D64, 287-301 (2008) (shameless plug) or you can add smaller PEGs or MPD which would probably help. The JAXA cryoprotectant database (http://idb.exst.jaxa.jp/db_data/protein/search-e.php) has many referenced entries for PEG 3350 and these may offer a clue. On a practical note cool the crystal across the thinnest axis, i.e. not from the tip to base and if possible (i.e. if the cross section is large enough for X-ray diffraction) harvest it before it gets too big - remember crystallography is all about reciprocal space; size is important, smaller is better. As an alternative and showing my old fashioned streak - with long rod like crystals just shoot them at room temperature and translate every so often. You can actually calculate the optimum translation distance but I'm not going there. The crystals will last a surprisingly long time at the synchrotron (see Southworth-Davies et al., Structure 2007, 1531-1541). You will have to play with capillaries or should I say 'caaapilllaries' for the American audience - I've never mastered that way to say it. Anyway, I've collected some very nice complete data sets with room temperature samples. With a 1 mm rod you are going to have quite a few points on the crystal to shoot ... brings back memories of the Laue days, dark rooms, stained shirts and that toast rack - sorry J :) Too much typing - I blame far to too much caffeine and lack of sleep after my daughter recently renamed all my laptop files to gibberish! Good luck, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! > On Sat, 14 Feb 2009 21:15:11 -0500, Keith Romano wrote >> Hi all, >> >> I have protein crystals in complex with substrate grown in 20-30% (w/ >> v) PEG 3350, 4% (w/v) ammonium sulfate, and 0.1M sodium MES buffer at >> pH 6.5. I purify and concentrate my protein in a high salt buffer >> (0.5M NaCl, 0.1M sodium MES at pH 6.5, 10% glycerol, 2mM DTT). >> >> I grow my crystals with vapor diffusion in 24-well format by hanging >> a drop of equal volume protein and precipitant solution over the >> reservoir of precipitant solution. Interestingly, when I do the >> math, the initial osmolarity of my drop is greater than that of the >> reservoir (due to the high NaCl in the protein solution). As far as >> I know, this runs against the principles of the vapor diffusion >> method, as vapor will leave the reservoir and enter the drop... >> >> Nevertheless, these conditions yield giant, rod-like crystals over >> 1mm long. However, they don't react well to direct flash freezing- >> the spots tend to smear and indexed refinement leads to high >> mosaicity. I have tried many cryoprotectant solutions by making up >> the given precipitant solution with 15-25% glycerol or ethylene >> glycol, including a range from 0mM to 350mM NaCl. In general, >> dipping the crystals in cryoprotectant improves the diffraction and >> lessens the "spot smearing". However, the diffraction usually >> becomes highly twinned and hard to index. After transfer into the >> cryoprotectant, the crysatls appear to crack and often break apart, >> as observed under the microscope. >> >> It seems like my crystals are very sensitive to the osmotic/ionic >> change when transferred to the cryoprotectant. I have been unable to >> find a "stable" cryoprotectant, and I am wondering if anyone has had >> similar experience with high-weight PEGs and could suggest some >> cryoprotectants to try out. >> >> Any input would be greatly appreciated! >> >> Keith >> >> Department of Biochemistry & Molecular Pharmacology >> 970L Lazare Research Building >> University of Massachusetts Medical School >> 364 Plantation Street >> Worcester, MA 01605
