Hi Xun, I'd find out what the NCS averaging correlation is between the molecules in the asymmetric unit. If your correlation is high then tight NCS should help in refinement. You could also take a look at the NCS averaged map in COOT. You can also have ideal helices build using COOT or PHENIX (find_helices_strands). This should give the ideal geometry. You can also refine with secondary structure restraints for proper hydrogen bonding patterns. If someone told you to focus on geometry, then I'd focus on the RMS bonds/angles and Ramachandran plot.
Best regards, -Reggie -- Reginald McNulty UC Irvine Department of Molecular Biology & Biochemistry Ph.D. Candidate On Jun 16, 2011, at 2:11 PM, Xun Lu wrote: > Hi, > > I have a 3.2A dataset for a protein-DNA complex. The protein is a > homodimer, and the DNA is almost palindromic (except one base pair in the > middle and two or three base pairs at both two ends). It is my first time > solving structures, and unfortunately the resolution is low. No body in our > lab has used ccp4 or phenix, so I am really frustrated as a second year > student. > I mainly used ccp4. So far, the best R/Rfree I got is 0.27/0.34. I > went to the crystallography meeting, and people suggested me to rely more on > geometry. I remember I got a DNA restraints file and a refmac script from > someone on this mailing list, and that really helped (otherwise the DNA base > pairing will be weird). Can someone tell me how to restraint the protein > (helix)? I can only see a few side chains in the helix, so it's hard to say > whether the registration of the helix is correct or not. Maybe my high R-free > is due to the helix in the wrong position? > People also suggested me to include NCS and TLS in the refinement, but > I don't know how to. For NCS, I should define a region that are the same in > both monomers? Should I use tight or loose restraints? For TLS, I don't have > a clue. > > I don't know whether I am doing the right thing. I did molecular > replacement (it seems I got the correct solution), then rigid body > refinement, then a couple of restrained refinement right after the rigid body > (why the R factors go down even that I haven't done anything to the > structure?), then I walked through the amino acids in coot and did whatever I > could. Actually since my DNA and protein helix are longer than the ones in > the model, so I built them myself. > > > Any suggestion is welcome! > > > Xun > > > > > -- > Department of Molecular and Structural Biochemistry > North Carolina State University
