I want to start off by thanking everyone. The replies, both on- and off-board, were speedy and numerous. I apologize for the delay in expressing my gratitude; I was implementing your wonderful suggestions. I have put together a summary for the archive.
To recap, I was looking for suggestions to cryoprotect crystals from >3 M NaCl crystallization conditions excluding ethylene glycol or PEG completely and avoiding glycerol and sucrose due to apparent crystal instability. -Several people confirmed that 4 M NaCl should be sufficient. -There were several suggestions for cryosalts with malonate and formate being the most frequent. Lithium salts were also put on the table including partial or complete substitution of NaCl with LiCl, both as a soak or a crystallization solution. -As an end run around the apparent glycerol instability, stepwise transfers and quick dips with glycerol were suggested. Glycerol supplemented with xylitol was also thrown into the mix. -There was one tale of sucrose being successfully added into a crystallization condition when direct soaks were unsuccessful -There were several confirmations regarding the success of Paratone and Paraffin oil with pro-tip of dehydrating the paraffin oil in a speed-vac overnight. -Additional suggestions included 50-75% saturated sugars and 6.5 M proline. Since I’m a good scientist I will include the reference section. I found it very useful. -Cryosalts: suppression of ice formation in macromolecular crystallography. K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 -Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. T. Holyoak et. al. *Acta Cryst.*(2003). D *59*, 2356-2358 -Proline: Mother Nature's cryoprotectant applied to protein crystallography. T.A. Pemberton et. al. Acta Cryst. (2012) D68, 1010-8. -Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov et.al. J. Appl. Cryst. (2006) 39, 244-251 -A comparison of salts for the crystallization of macromolecules. A. McPherson. Protein Sci. (2001) 10, 418-22 -Strategies for protein cryocrystallography. L. Vera, E. A. Stura. Crystal Growth & Design (2013) 14(2), 427-435 Thank you all again. I really appreciate your time and energy. Cheers, Katherine On Wed, Feb 19, 2014 at 9:23 AM, Enrico Stura <est...@cea.fr> wrote: > Dear All, > > I would like to point out that the conditions 1.8 - 2.0 M NaCl are not > considered High Salt as > NaCl is soluble to 5M and a 2X solution (i.e. 4M NaCl) is possible. Also > NaCl contrary to > ammonium sulfate, citrate, phosphate, etc. is compatible with polyethylene > glycol without phase > separation problems. > > This means that with 1.8 - 2.0 M NaCl you have an vast repertoire of > possible ways > to cryo-protect crystals and with the vast repertoire you gain a good > possibility of finding > conditions that enhance diffraction: > see: > Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. > Crystal Growth & Design, > http://pubs.acs.org/doi/full/10.1021/cg301531f > > For me the definition for "High Salt" is that 2X for the precipitant > component is not possible. > > Enrico. > > > On Wed, 19 Feb 2014 16:06:27 +0100, Karolina Michalska <dzi...@amu.edu.pl> >> wrote: >> > > >> 4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M >> NaCl. >> >> Karolina >> >> W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a): >> >> 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- [1] >>> >>> X-ray lab webpage: http://www.oumedicine.com/department-of-biochemistry- >>> and-molecular-biology/department-facilities/macromolecular- >>> crystallography-laboratory [2] >>> >>> Small Angle Scattering webpage: http://www.oumedicine.com/ >>> docs/default-source/ad-biochemistry-workfiles/small- >>> angle-scattering-links.html?sfvrsn=0 [3] >>> >>> ________________________________________ >>> 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=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4 >>> V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF% >>> 2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3d >>> d6e2ded91923c614e670857e10916c687e [4]> 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 >>> >> >> >> Links: >> ------ >> [1] >> http://www.oumedicine.com/department-of-biochemistry- >> and-molecular-biology/faculty/blaine-mooers-ph-d- >> [2] >> http://www.oumedicine.com/department-of-biochemistry- >> and-molecular-biology/department-facilities/macromolecular- >> crystallography-laboratory >> [3] >> http://www.oumedicine.com/docs/default-source/ad- >> biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0 >> [4] >> https://urldefense.proofpoint.com/v1/url?u=http://archive.com&k= >> 7DHVT22D9IhC0F3WohFMBA%3D%3D%0A&r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4 >> V0aIwH4RJhyZU%3D%0A&m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF% >> 2FzyGkSwsbIoq92CSnOk%3D%0A&s=3cfbf18821b5b59934971bf583cf3d >> d6e2ded91923c614e670857e10916c687e<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> >> > > > -- > 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 > -- "Nil illegitimo carborundum"* - *Didactylos
