Hi Ngo, Obviously, you can spend years optimizing crystallization conditions for a membrane protein. Or, you may get lucky and do it quickly. In any case, I would recommend to try, in parallel, collecting data on one of the micro-diffraction beamlines. We offer such capability at GM/CA-CAT beamlines at APS (http://www.gmca.anl.gov/ <http://www.gmca.anl.gov/> ) and have had some good success with it (e.g. Nature, 2007 Nov 15;450(7168):383-7, Science, 2007 Nov 23;318(5854):1258-65, Acta Cryst. (2008). D64, 425-435). There are few more such facilities in the US and few more in Europe. Can you tell what is the size of your needles in cross-section? Good luck! Nukri
Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT, Bld. 436, D007 Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> ________________________________ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Van Den Berg, Bert Sent: Wednesday, April 16, 2008 9:09 AM To: [email protected] Subject: Re: [ccp4bb] Optimization of needle crystals? Hi Ngo, your needles actually look like very thin plates. They seem promising to me. From appearance its impossible to tell whether the crystals are detergent or not. DDM is apparently known to form crystals, but I've never really gotten any (DDM is very soluble). What temperature have you used? 4C would favor forming DDM crystals. The round crystals are often seen in membrane protein crystallization. They generally don't diffract, contain a lot of detergent (maybe all detergent) and are very hard to get better (ie get sharp edges and/or diffraction). What I would do at this point is trying to get the needle/plate crystals better (by using techniques/tricks that are used for soluble proteins, such as vary Mg2+ salt conc and identity, temperature, volume/reservoir ratio, protein conc. and even trying different detergents and/or mixtures including DDM). For now I would go with the assumption that the crystals are protein. Good luck! Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm ________________________________ From: CCP4 bulletin board on behalf of Ngo Duc Tri Sent: Wed 4/16/2008 9:48 AM To: [email protected] Subject: Re: [ccp4bb] Optimization of needle crystals? Dear experts, You didn't mention which detergent in the conditions. From the picture it looks like crystals of detergent. The initial buffer contains 50mM Tris 7.7, 100mM NaCl and 0.015% DDM. I got two forms of crystal: needle and round shape. The needle crystals grow from conditions containing Mg2+ salt and high pH. The round crystal grow from conditions containing Mg2+, high pH and small PEG. I already fail to optimize the round shape (by changing the PEG concentration as well as pH or protein concentration). So that's the reason why I think about optimization of the needle form. Here I attach more pictures to show you the needle and the round crystal. I never applied powder diffraction before so maybe it's hard for me to confirm it's the crystal of detergent or not. Thank you very much for all of your helpful suggestion! My best regards, TriNgo PhD Student- Sungkyunkwan University, Korea
