Hi Mary, There are many excellent suggestions already but you may want to try tweaking your cryo a bit. This is described very nicely by Ed Mitchell and Elspeth Garman in a paper on mosaic spread as a function of cryoprotectants concentration - J. Appl. Cryst. (1994) 27, 1070-1074. You already have a cryoprotectant, the MPD, and could up the concentration a little by successive transfer or adding a little to the drop. I've also found that (2R,3R)-(-)-2,3-butanediol works as a 'magic' elixir in very small concentrations when added directly to the drop.
I'd say try room temperature but at that resolution you are going to need all the X-rays you can get onto it. Cheers, 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: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! Mary, freezing habitually increases mosaicity. In your case, the high water content adds to the problem. Try not to freeze the crystal but collect at sub-zero temperature (in short glass capillaries or use oil plugs instead). You have to optimize the "close to freezing" data-collection temperature. I collected complete synchrotron datasets (of GCPII in buffer with PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity values of as small as 0.07 degrees! At 277 K, the crystals only last for a few images and freezing did not work (for the buffer mentioned before). - J. - > -----Original Message----- > From: Mary Fitzgerald <[EMAIL PROTECTED]> > Date: Mon, 9 Jul 2007 18:05:10 > To:[email protected] > Subject: [ccp4bb] Help with reducing crystal mosaicity > > Help please! > > I'm looking for some new ideas. I have crystals that come out of a > sitting drop with a mixture of sodium cacodylate at pH 6.5, magnesium > acetate and MPD for the well solution. The MPD concentration is > sufficient to act as a cryoprotectant. Currently, I directly freeze > these crystals in liquid nitrogen. When I collect data, I typically > have high anisotropic mosaicity; it ranges from 0.8 to 1.2. This is > further complicated with a weakly diffracting crystal (4-5 A) that has > a long unit cell axis of ~500 and often twinning. > > It has been suggested to me that the cryoprotectent is a problem. I > haven't checked the diffraction at room temperature, yet. Please no > suggestions of finding a different crystal form as that's not a > consideration at the moment. I have my reasons. I did find one > crystal that has lower mosaicity (0.5 to 0.8) but had weaker > diffraction then the typical crystal. Attempts at flash cryoannealing > have not helped. > > So, what's a good way to change the cryoprotectant if the > cryoprotectant is the precipitant? I've considered trying dehydration > but wasn't certain if that would help with the mosaicity. > > Thanks for any ideas, > > Mary X. Fitzgerald > Postdoctoral Associate > -- Jeroen Raymundus Mesters, Ph.D. Institut fuer Biochemie, Universitaet zu Luebeck Zentrum fuer Medizinische Struktur und Zellbiologie Ratzeburger Allee 160, D-23538 Luebeck Tel: +49-451-5004070, Fax: +49-451-5004068 E-mail: [EMAIL PROTECTED] Http://www.biochem.uni-luebeck.de Http://www.iobcr.org Http://www.opticryst.org -- If you can look into the seeds of time and say which grain will grow and which will not - speak then to me (Macbeth) --
