Seeding! Make seeds, rescreen with seeds. Look in many former ccp4bb posts for references about this. Jacob
----- Original Message ----- From: Jürgen Bosch To: [email protected] Sent: Tuesday, October 26, 2010 3:47 PM Subject: Re: [ccp4bb] Help with Optimizing Crystals You did check on a gel that they are indeed your protein ? If you have sufficient amounts available try digesting it with various proteases and see if you can identify a stable fragment. A less radical approach, which might not be accessible to you, you could screen your protein for alternative buffer conditions using DSF and then pick a condition under which it seems to be very stable according to its melting temperature in the buffer. You've spared us the details of your purification procedure, maybe a polishing step at the end with a SEC might do wonders. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 26, 2010, at 4:23 PM, Matthew Bratkowski wrote: Hello. I have obtained disk shaped crystals of a protein that I am working on. I got hits in about 10 different conditions, with a few common precipitants and pHs, and I have optimized two conditions so far. In the optimized conditions, the crystals appear overnight, usually surrounded by or hiding under heavy precipitant. Under the best conditions, I get what I would describe as single disks, some of which are of decent size and very round, that rotate light very well. Sub-optimal conditions can give small to large crystal clusters. I shot the large disk crystals grown from one conditions at the synchrotron. but they do not diffract. I was wondering if anyone had any advice about optimizing these crystals in order to get them to diffract better? As mentioned before, I have only tried optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), but crystals from all of the hits look the same: always round disks or disk clusters. This leads me to believe that optimized conditions of the other hits will produce similar results as before. Would it be worthwhile to try optimizing these conditions as well? I have also tried seeding, which just produces a lot of clusters, and an additive screen. Some of the additives help to produce larger crystals, but again I always get single or disk clusters. Any advice would be helpful. Thanks, Matt ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [email protected] *******************************************
