Another thing you should bookmark and check out is the Molprobity page (google for it), upload your pdb file and let it evaluate what you have done. The chart it generates will guide you through your structure and you will be able to do a good job.
Jürgen

......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/

On Jun 17, 2011, at 8:30, Robbie Joosten <[log in to unmask]> wrote:

> Dear Xun,
>
> > > 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.
> Indeed, this is just asking for problems. It's a good call that you asked for help. Perhaps your supervisor can arrage for you to be embedded in a crystallography lab for a while. That should give you easy access to people with experience.
>
> > > 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
> You are heading the right way. You should be able to close the R/R-free gap a bit more.
>
> > > 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 like real-space refining everything in Coot with tight helical restraints. You may need to chainge the default restraint weight matrix (lower numbers give tighter restraints). The options are under the "R/RC" button.
>
> > > 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.
> In Refmac you can use "local NCS" which takes away the need to mess with NCS selections (which can be really difficult). Although it is not needed for Refmac, you should make sure that the same residues in different monomers have the same residue number. Be conservative with TLS (in the beginning). One group per chain sounds right. In the case of your DNA you can consider putting both chains in one group. Tight B-factor weights may be needed, you could also trying one overall B factor. I personally only do that when TLS works well. Oh, and always use riding hydrogens in refinement. It helps a lot at low resolution, because of the VDW restraints. For that same reason you should not be too conservative with the sidechains (at least not for the ones in the core of the protein).
>
> Since you have only started building you should probably go through the entire structure a few times. After that, use structure validation tools frequently. WHAT_CHECK and Molprobity are must-use tools for that. Coot also has many usefull features for validation. Good luck.
>
> Cheers,
> Robbie Joosten