Hi,
1. You can learn about compound binding using methods orthogonal to X-ray (i.e. SPR, NMR, and so forth). If the sole detection method available to you is X-ray then you should expect to need a reasonably complete dataset in order to determine binding - otherwise you can easily miss binding of a weak ligand. If you countersoak against a co-crystallized colored or fluorescent ligand you may detect exchange visually however it's not fool-proof either. 2. Most library screens done by X-ray are done in multiplex. Typical mixtures are anywhere from 4 to 8 compounds per soak. Resolution of individual hits is sometimes non-trivial as compounds may have unpredictable synergies with respect to binding. As a separate note - if *most* of your inhibitors seem to inhibit crystallization of an otherwise 'easy' protein - perhaps the issue is not in the compounds themselves but in the solvent (commonly DMSO) that they're in. Have you found out if DMSO alone (or whatever solvent you're using) inhibits crystallization? This isn't the only option but it's a fairly common one. Artem _____ From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Rongjin Guan Sent: Thursday, November 12, 2009 3:57 PM To: [email protected] Subject: [ccp4bb] small molecule soaking screening Hi All Sorry that this is a non-ccp4 question, but I hope I can get some good suggestions from the community. We have protein crystals under various conditons and want to soak them with different potential inhibitors. Most of inhibitors have very small molecular weights (200-300), so it become a problem how to detect if the small compounds have been soaked into the crystals or not. (co-crystallization experiments yielded no hits so far, though the free form is easy to be crysatllized under many conditions) We pay $500/day for local X-ray facility access, so we wonder if there are some more efficient ways that allow us to know if the small compounds soaked in or not, without collecting a whole data set for MR. We are also thinking if we can mix several compounds together for soaking, to reduce the combinations of soaking experiments with various compounds and crystals from various conditions. Is this practical, if some of them have pretty similar binding affinities to the protein? All comments/suggestions are welcome. Thank you Rongjin Guan
