On Thu, Jun 16, 2011 at 2:11 PM, Xun Lu <xlun...@gmail.com> wrote:
> 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?
>
Refmac uses a syntax like this to define custom restraints (apologies to
Garib if I'm screwing this up):
exte dist first chain B residue 5 atom N second chain B residue 1 atom
O value 2.900 sigma 0.05
Phenix can generate the annotations for you - either automatically if you
run phenix.refine, or by doing this:
phenix.secondary_structure_restraints model.pdb format=refmac
The high R-free could be due to any number of problems, but the structure is
obviously a little bit overfit (that is, the difference in R-work and R-free
is too large); adding NCS restraints and TLS usually helps this, sometimes a
lot. You may have an easier time interpreting sidechain density if you try
B-factor sharpening the maps, which both Refmac and Phenix (in addition to
CNS) can now do.
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.
>
For TLS, you could start with one group per chain. You can get a more
fine-grained division by running the TLSMD web server (
http://skuld.bmsc.washington.edu/~tlsmd/) or phenix.find_tls_groups.
NCS restraints are a little trickier and others will probably have better
advice for you - however, if there is NCS present, you should absolutely use
these restraints at 3.2A resolution.
-Nat
