I suspect if you compare Ramachandran plots you'll find that despite the slightly higher Rfree, the ncs-restrained coordinates already have better geometry.
You probably need to optimize your ncs restraints. For example: - remove the restraints all side chains that are in different crystal packing environments on different monomers - if your protein has more than one domain, they might be in slightly different relative positions in different monomers, in which case they should be restrained separately - remove the restraints for any obviously-variable surface loops One way to find the problem areas is to superimpose coordinates refined with and without the ncs restraints, and look for interesting differences. A more quantitative way to do it is to plot the difference in real-space R for each residue (after refining both ways), and look for spikes. I don't know how to do that with ccp4 but you can certainly do it by running the real-space R routine in cns, then plugging the output into excell. Good luck! Phoebe ---- Original message ---- >Date: Tue, 29 Apr 2008 14:02:22 -0700 >From: Kianoush <[EMAIL PROTECTED]> >Subject: [ccp4bb] Low resolution structure refinement >To: CCP4BB@JISCMAIL.AC.UK > > Dear All, > > I have a protein structure I am trying to refine > using Refmac. There are ~158,000 atoms modeled (no > waters) in the asymetric unit with ~495,000 > reflections. There is data to 3.0A resolution with > the 2 sigma cutoff at 3.5A. I have four NCS copies > in my asymetric unit and when I specify NCS > restraints, my Rfree gets slightly worst (from 28% > to 31%). Varying the weighting term has not made a > difference and the Rfree is generally constant > between refinement cycles. My questions are: > > 1) For an all-atom refinement my observation to > parameter is ~1.3. What is the most suitable > refinement method for this scenario? Is there a way > to find out roughly my observation:parameter ratio > based on the type of refinement I choose? How much > are the restraints adding to my observations in the > ratio? > > 2) It makes sense that NCS restraints should help > the refinement, but it is not as judged by the > Rfree, or is the 28-31% difference acceptable at > this resolution? > > 3) I have the same sort of problem when including > phases in the refinement. > > Thank you, > Kianoush > > ********************* > Kianoush Sadre-Bazzaz > Graduate Student, Chris Hill Lab > University of Utah Biochemistry Department > 15 N Medical Drive East RM 4100 > Salt Lake City UT 84112-5650 USA > (801)585-7068 > [EMAIL PROTECTED] > > > > ------------------------------------------------ > > Be a better friend, newshound, and know-it-all with > Yahoo! Mobile. Try it now. Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
