Roger's note reminded me of some older literature (old in the sense that this problem extends back into the mid-1970's). Dealing with cryopreservation of crystals grown in "high" salt can be a real problem, but as many people have pointed out, the normal cryoprotectants can work, although many salts work just as well (not only malonate, but ammonium formate, lithium citrate, etc.). I don't consider 1.6-2 M ammonium sulfate as really high salt; try working with 3 M ammonium sulfate. However, the trick is not simply the cryoprotectant, but also the exchange method, as Roger mentions. While you can make artificial mother liquors (I love these old terms) with high concentrations of salt and nonionic cryoprotectants (sugars, glycerol, ethylene glycol, etc.), that does not mean they will readily exchange with the crystal, even after soaking for hours.
Bill Ray, a classical enzymologist and physical biochemist from Purdue, really wanted to determine the crystal structure of phosphoglucomutase with ligands, but there were many difficulties. Using crystals grown in 2.1 M ammonium sulfate was one of these problems. He realized that the phase interactions between the bulk solution (i.e., the mother liquor) and the interstitial salt-rich solvent was a major obstacle in the proper solvent exchange and good subsequent cryopreservation. See how he solved the problem: W. J. Ray, Jr., et al. Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by X-ray crystallography at low temperature. Biochemistry. 1991 Jul 16;30(28):6866-75. Cheers, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: rmgarav...@gmail.com **************************************************************** On Jul 12, 2012, at 2:10 PM, Roger Rowlett wrote: > We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M > ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% > glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium > sulfate (5.6 M) have enormous solubilities in water, so I would not expect > cryoprotectant concentrations of glycerol or glucose to cause precipitation > (We can save cryoprotectant solutions of at least 2 M ammonium sulfate > indefinitely). How are you introducing cryprotectant? We use one of two > methods: > > Fish the crystal out of the mother liquor and place into artificial mother > liquor with the same composition as the well solution + cryoprotectant. For > glycerol or other liquids, you have to make this from scratch. For glucose, > we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the > 1.0 mL mark with well solution. (Mix well of course before use. Gentle > heating in a block or sonication will help dissolve the glucose. > Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop > the crystals are in. You can do this all at once, or in stages, keeping the > drop hydrated by placing the hanging drop back in the well between additions. > If your drops are drying out during crystal harvesting (very possible in dry > conditions), you might try harvesting in the cold room, where evaporation is > slower. We often have problems with crystal cracking and drop-drying in the > winter months when the humidity is very low indoors. The cold room is usually > humid enough and cold enough to slow evaporation to allow crystal harvesting. > (I hate working in the meat locker, though.) > Cheers, > _______________________________________ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > > On 7/12/2012 12:55 PM, m zhang wrote: >> Hi Jim, >> >> 25% is w/v. Thanks for the information. Will check the webinar. >> >> Thanks, >> Min >> >> From: jim.pflugr...@rigaku.com >> To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK >> Subject: RE: [ccp4bb] cryo for high salt crystal >> Date: Tue, 10 Jul 2012 17:39:56 +0000 >> >> Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v >> or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, >> or 50% saturated in reservoir. You will have to TEST these. See also this >> webinar on cryocrystallography which shows how to make these solutions: >> http://www.rigaku.com/node/1388 >> >> You could also try high salt solutions with similar technique. >> >> Good luck! >> >> Jim >> >> >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang >> [mzhang...@hotmail.com] >> Sent: Tuesday, July 10, 2012 11:28 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] cryo for high salt crystal >> >> regaentDear All, >> >> I am sure this question was discussed before. But I am wondering if anyone >> got the same experience as I do. >> I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. >> I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or >> ammonium sulfate itself: The problem is that all the cryo plus original >> reagents in the reservoir precipitate the salts out. And more serious >> problem is because of high salt in the condition, while I am trying to loop >> the crystal, both the drop and >> cryoprotectant drop form salt crystals (not sure it is KCl or ammonia >> sulfate) significantly and very quickly, that cause my crystal dissolved. My >> crystal doesn't seem to survive paraton-N oil. Does anyone here have >> similiar case? any suggestion will be appreciated. >> >> Thanks, >> Min > >
