For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4 uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4 M. We do not bother withdrawing aliquots to maintain a fixed volume. You may need to tweak the volumes to optimize the resulting diffraction. You can also break the additions at given concentration into smaller aliquots to reduce the osmotic shock. This approach is much gentler than transferring the crystal directly to 3 M sodium malonate. Do not leave the drop exposed to the air for more than 3 minutes or so because salt crystals will start to grow. When there are multiple crystals in a drop, often the unused crystals in the very high salt solution will still diffract well up to a year later if the crystallization chamber is resealed well; their diffraction might even improve with the prolonged exposure to high salt.
Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK 73104-5419 Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190 office: (405) 271-8300 lab: (405) 271-8313 fax: (405) 271-3910 e-mail: blaine-moo...@ouhsc.edu Faculty webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d- X-ray lab webpage: http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory Small Angle Scattering webpage: http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0 ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katherine Sippel [katherine.sip...@gmail.com] Sent: Tuesday, February 18, 2014 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] High Salt Cryo Hi all, I'm looking for a cryo condition for high NaCl (3+ M) crystallization condition. I would do it the proper way, but our beam/cryostream is down. I've tried a bunch of things at the moment. Ethylene glycol and PEG 400 nuke the crystals immediately even at low concentrations. Prolonged exposure to glycerol and sucrose starts to break them down so I'm thinking that the diffraction will probably suffer. I can't find any reports of NaCl's viability as a cryosalt. I've got Paratone/Paraffin oil/Mitegen's LV cryo oil on tap but I was hoping to not put all my eggs in one basket. I tried the ISRDB database through archive.com<https://urldefense.proofpoint.com/v1/url?u=http://archive.com&k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0A&r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e> without any luck (no search function). I've gone to the PDB searching for similar crystallization conditions and looked up the papers for their cryos, but they are all glycerol. Google gives me the same. I thought I'd see if anyone on the bb has an anecdotal "this worked for us" story. I would love to hear it. Thank you for your time, Katherine -- "Nil illegitimo carborundum" - Didactylos
