Dear Frank, Toufic is right. The trick is no trick, you just have to try everything. In my experience if u have a protein crystals in a mild detergent and it is very fragile no point going to a harsh or shorter chain detergent like OG. I got crystals in NM (9 C) and they were fragile and dissolved after 1 day. I could only get stable crystals when I moved to DM (10 C) and DDM (12 C). Once you get a stable crystal you may consider dehydrating them in higher PEG so as to improve protein protein contact. Again the trick is no trick, you just try everything suggested. Good luck with your trials. Emmanuel Nji.
On 23 October 2013 19:46, El Arnaout, Toufic <elarnao...@biochem.wustl.edu>wrote: > Hi Frank, > The previous suggestions are great. In my case, I had soft crystals (bend > from 180° to ~130-140°) in the loop using the LCP but they still > diffracted. There are a lot of "heroic stories" on how some people solved a > structure, you should just try as many ways as possible (do not only rely > for a long time on only one condition or construct), and remember "methods" > are just methods (i.e., there are hundreds of LCP robots in the world..). > Protein engineering, homologs (maybe hyperthermophiles).. stabilization.. > are great ways. > Recently I had tested various crystals grown by vapor diffusion for a > protein purified in different ways/detergents, so obtaining crystals is > very common, but it's just the start. I also had crystals using bicelles > but saw huge spots to 2 A (no patterns.. rather looked like > detergent/lipid). > Good luck > > toufic el arnaout > > > > > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of crystalboy > [yyb...@gmail.com] > Sent: Wednesday, October 23, 2013 11:22 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] membrane protein optimization > > Hi CCP4BB Forks, > > In recently I got a membrane protein crystal in the quite normal > membrane protein crystallization conditions as other persons reported, > like PEG400 16-19%, pH 6.0-7.5, with 50 mM MgCl2 (in my case) by using > sitting drop method. These crystals are around 50-100 uM. They look > like trapezoid crystal. My problem is all of my crystals have not > diffraction in home source X-ray and just poor diffraction at > Synchrotron (lower than 20 A). My crystals like to appear on the > surface of the drop. Look like my crystals are quite light. I had > tried to use a needle to touch them. Unlike other protein crystal, my > crystal looks like quite "soft". When I touch it, it didn't crack, but > was bend or mashed. I had tried to do additive screen and detergent > screen. It seems they are not useful. > > Do anyone have good ideas to optimization these crystals? Thanks for > your suggestions. > > Frank > > -- Dr. Emmanuel Nji Department of Biochemistry and Biophysics Centre for Biomembrane Research Stockholm University 106 91 Stockholm Sweden Cell: 0046736457727
