Hi Matt, You'll probably get many different answers to a question like this, but what I would do is go back to your protein and make different constructs; chop off termini, surface mutations etc, maybe cleave off the tag. Of course more screening and optimization might work, but my sense is that since you get many hits pretty easily that however don't diffract, there may be something on the protein level that needs correcting.
Good luck, Bert On 10/26/10 4:23 PM, "Matthew Bratkowski" <[email protected]> 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
