Hi Nian, This isn't a solution but I remember seeing an excellent talk by Annie Hassell at the CCP4 study weekend a couple of years back.
The paper from that talk may help you out. Acta Cryst. (2007). D63, 72-79 [ doi:10.1107/S0907444906047020 ] Crystallization of protein-ligand complexes A. M. Hassell, G. An, R. K. Bledsoe, J. M. Bynum, H. L. Carter, S.-J.J. Deng, R. T. Gampe, T. E. Grisard, K. P. Madauss, R. T. Nolte, W. J. Rocque, L. Wang, K. L. Weaver, S. P. Williams, G. B. Wisely, R. Xu and L. M. Shewchuk Synopsis: Methods presented for growing protein-ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals. HTH, Dave On 12/09/2007, Joe Krahn <[EMAIL PROTECTED]> wrote: > I am working with a protein/ligand complex that is very resistant. to > getting a stable complex. The first thing to try is to go to saturating > ligand concentrations, even if it seems that it is excessive. It is > possible that binding affinity is significantly different. > > My biggest problem is precipitant interfering with binding, combined > with relatively low substrate solubility. I tried transferring to a > different condition, but it seems that most precipitating agents have a > solubility affect on the ligand as well. I ended up screening conditions > just to understand ligand solubilities. I even tried things like DMSO > additives. > > Eventually, I found conditions where the ligand is fairly soluble, but > also stabilizes the protein. I found that ethylene glycol helped binding > even though glycerol inhibited it. I also changed the pH, and did the > soak at room temperature instead of in the cold room. Even then, I used > saturating ligand concentrations. > > Joe Krahn > > Nian Huang wrote: > > Dear All, > > > > I have been trying to get a protein-ligand complex crystal for a long > > time. Here, ligand is either substrate or product for this kinase. I > > have tried many methods: soaking with apo-crystal, co-crystal with > > different concentration of ligand and screen for new conditions. I got > > a couple of co-crystals with the new conditions, but still no ligand > > was found in the active site. > > > > Anybody has some new ideas that I can try? I have been using glycerol > > as my cryoprotectant. Could cryoprotectant have some negative effect > > on co-crystal? I will try to collect some data at room temperature in > > the future. > > > > Thank you in advance for your suggestion. > > > > Nian > > > > Dept of Biochemistry > > UT Southwestern Medical Center > -- --------------------------------------- David Briggs, PhD. Father & Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --------------------------------------- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams
