Dear all,
I am working on a structure which is composed of two domains. The X-ray data is quite good (P21, 2.2A, Rmerge=0.08, 93% completeness), there is one molecule (277 residues) per ASU. The structure has four homologous structures with sequence identities ranging from 35%-40%. The four structures superposed quite well and there is no relative movement between the two domains. However, MR with these probes all failed using Molrep or Phaser (R/Rfree after refinement is 51%/55%, and the density is poor). Then I searched the two domains separately and located the first one (about 60% of the structure, 44% identity) unambiguously but failed to find the second one (36% identity) either by searching it alone or fixing domain 1. The R and Rfree after refining domain 1 are 39% and 44%. The density is good for the existing model but disconnected in the missing part of the structure in which I can hardly place any residue. Only an obscure shape of beta sheet could be recognized but its position relative to domain 1 is quite different from those in the homologous structures. Thus I suspect that domain 2 has undergone movement or adopted a distinctive conformation comparing to the known structures. I searched the CCP4BB archive and found a similar case from Eric Liu posted in the end of July this year. The suggestions are to mask the whole molecule and perform DM, or to carry out OASIS-DM-Building iterative cycles (as published in Acta Cryst. (2007). D63, 793-799), or to measure the experimental phases, and etc. So I tried to make a solvent mask in coot 0.3.3 using the "mask map by molecule" function. I read in the map (from refining the first barrel) and the superposed complete homologous structure, masked the map by it, and exported the masked map. Then I used MAPMASK utility to convert the map to mask (the density cutoff is set to 1) and feed it to DM. But the resulted MTZ contained all zero reflections. Thus my questions are: 1) How can I make a mask as mentioned above? Am I doing it the wrong way? 2) Is there any other method to improve the density of the missing structure? I tried several MR probes of the second barrel fixing the first one but all failed. Sorry for the naive question, but this has bothered me for quite some time. Any suggestions will be greatly appreciated. Thanks in advance! Tiancen Hu Shanghai Institute of Materia Medica Chinese Academy of Sciences
