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 resolutionYou 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
