Hi Have you checked for twinning?
J TC Hu <[EMAIL PROTECTED]> wrote:> > 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 -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
