we were also once able to change 1.7 M Amm sulphate to about equal ionic strength of citrate, in a few concentration steps of mixtures (for other reasons, it was supposedly an inhibitor..).you probably can switch to some friendlier salt... plus you could combine that with gentle crosslinking through vapour diffusion first, if needed etc. (although try without first.)
t. Quoting [EMAIL PROTECTED]: > Tiancen, > > How critical is it that the crystals remain in the sulfate salts? I ask > because we recently had success with an RNA crystal that was very > intractable to heavy atom soaks because of very high lithium sulfate > growth conditions, and the fact that cryo-protection was best done by > increasing the lithium sulfate to near saturation. At that high sulfate > concentration, many of our favorite heavy atoms (mostly divalent and > trivalent cations since the crystals were RNA) are insoluble, and the > high Li+ concentration competed off the heavy atoms. > > What I did was move the crystals, in steps, from the 2M lithium sulfate > into high concentration of lithium Acetate. What this did was increase > the solubility of the heavy atom cations, which still maintaining high > ionic strength and offering cryoprotection. Ultimately, what worked was > 3M lithium acetate + 100 mM cobalt hexammine or iridium hexammine (yes, > 100 mM trivalent!). We got a nice derivative, and in fact the > diffraction limit of the crystal increased by almost 0.5 Angstroms! > > This was with an RNA crystal, so your problems may be different, but > perhaps something analogous might work, or at least you can get some > ideas from our experience. > > In short, if solubility of the heavy atoms is an issue, you may be able > to move the crystals into an alternate high salt condition that will > give better solubility, and then crank the heavy atom concentration up > to compete with the salt. > > Good luck, > > Jeff > > _______________________________________ > Jeffrey S. Kieft, Ph.D. > Assistant Professor > Dept. of Biochemistry and Molecular Genetics > University of Colorado School of Medicine > > http://www.uchsc.edu/sm/bbgn/kieftj.htm > http://www.evolutionarygenomics.com/CERT/CERT.html > _____________________________________ > For mail: > UCHSC at Fitzsimons > Mail Stop 8101, PO Box 6511 > Aurora, CO 80045 > > For courier/packages: > South Building RC-1, Room 9110 > 12801 East 17th Ave. > Aurora, CO 80010 > > phone: 303-724-3257 > fax: 303-724-3215 > email: [EMAIL PROTECTED] > > "Open your eyes. You have only to see things clearly, to understand." > -Leonardo da Vinci > > -----Original Message----- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of > Tiancen Hu > Sent: Thursday, April 19, 2007 10:27 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] recommendation for heavy atoms used in lithium sulfate > and ammonium sulfate > > Dear all, > > Could any one recommend some heavy atoms used for crystals grown in 0.1M > tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? > I read from Hampton user guide of heavy atom kit that "high salt > concentrations are not the ideal medium for heavy atom reactions with > macromolecules", so is there any type of heavy atom we should avoid > using? We do not have any experience in preparing heavy atom derivative, > so any suggestions, experience or references will be greatly > appreciated. > > Thanks in advance! > > Tiancen Hu > Shanghai Institute of Materia Medica > -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology PO box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
