> 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.
Your frustration is understandable. It is somewhat of an expectation in academia that your advisor will either help you directly or if she/he is not familiar with the methodology you are forced to use, will find someone to help you. The questions you ask surely may be answered by someone in your department. IMHO, a second year student should not be left alone to battle his first structure which happens to be 3.2A protein/DNA complex. > I mainly used ccp4. So far, the best R/Rfree I got is 0.27/0.34. and that is not bad given the resolution > 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)? one way of doing it would be to restrain the hydrogen bonds that stabilize the helix. It is not advisable at higher resolution, but sounds alright at 3.2A. I once used a restraint file to keep DNA sane by forcing Watson-Crick pairing, the helical restraints would work pretty mnuch in the same way. Look at the structure of the restraint file that you have and modify it to include the helix-stabilizing hydrogen bonds. > 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? It's not too high given the resolution. Also, at this resolution many sidechains will indeed appear missing, you'd have to use your best judgement to model things properly. Again, it's best if you can get an experienced "body" to sit down with you in front of a computer and look at your model. > 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. Yes and tight (at least at first). For TLS you may want to take a look at the TLSMD server. (Also, consider tighter restraints on B-factors). Otherwise, just define TLS for the whole thing, then protein and DNA separately, then individual monomers and whatever pieces of DNA common sense suggests would move together. Keep whatever combination gives you the lowest Rfree. > I don't know whether I am doing the right thing. Given the R/Rfree, you are on the right track. > 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?), because refmac have done something to it > 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. This http://xray.bmc.uu.se/gerard/supmat/rfree2000/plotter.html may make you feel better. I think an important question for you to ask is what is the structure telling you at this point. You already know in overall the architecture of your complex. At this data quality you may be able to figure out a little more regarding specific protein-DNA interactions (i.e. which residues define target recognition). Frankly, you can't expect much more. Cheers, Ed. -- "Hurry up before we all come back to our senses!" Julian, King of Lemurs
