Peter, The R/Rfree values seem reasonable given the resolution of the data and the completeness of the model. If you are unable to improve the model I would suggest getting an experimentally phased solution MAD or MIRAS. If your starting MR model is too incomplete you may not be able to get past the initial modal-bias. However, one method which I have used successfully on several structures is to use DM (RESOLVE SOLOMON etc.) on the model-phased maps. The improvement with 2-fold NCS is minimal, but the solvent-flattening will help significantly and may give clear density where you previously had only noise. If the map is improved then the autobuild feature in RESOLVE or TEXTAL will aid in building a better starting model for further refinement.
A script for using RESOLVE to generate maps is part of the PMB package for CNS (http://xray.utmb.edu/PMB/). It also requires some CCP4 programs for generation of the MTZ. There are also some examples on the RESOLVE homepage. Mark On Thu, 2007-02-15 at 12:12 -0500, Peter J Stogios wrote: > Hi, > > Long-time reader, first-time writer to ccp4bb. > > I'm having a problem with a 3.0 angstrom dataset (2.8 if I push it). > These crystals were thin but very long needles and I could see > diffraction only at the synchrotron. Diffraction spots were very > very small but well defined. High level of radiation damage. P2(1)2 > (1)2(1), no large unit cell axes. R-int 7%(14 in highest shell), I/ > sigma 14(8), completeness 98%. Nothing weird so far and the stats > actually look good. I was ecstatic these tiny needles diffracted at > all! > > I expect 2 chains by Matthew's coefficient and 50% solvent. > Biological unit is a dimer, homologs are dimers, this protein > purifies as a dimer. I'm solving it by molecular replacement, using > Phaser, which will give a refine-able solution only when searching > with dimeric models from homologous proteins. I cannot get solutions > that make sense or give Z-scores higher than 5 when searching for two > chains. But that doesn't matter, I get a solution when I search for > a dimer that is able to refine. > > So far so good. Here's where I get stuck: refinement with Refmac > goes well until R/Rfree values of 32/35, but I cannot break this > barrier. In fact, building into positive Fo-Fc peaks results in R- > free getting worse--actually any further refinement at all results in > R-free going up. The model is only 40% complete and has many missing > regions, not only in loops in turns. I just can't improve the model > anymore. I've tried rebuilding in resolve, Arp/warp, and of course > lots of manual building. > > I don't think my data is as bad as to restrict my refinement, so I'm > confused as to why I've hit this barrier. Hopefully this description > isn't too vague but I appreciate any help in advance and I can > elaborate if you're willing to help!! > > > ~ > Peter J Stogios > Ph.D. candidate, Privé Lab > Dept. of Medical Biophysics, University of Toronto > Toronto Medical Discoveries Tower (TMDT) at MaRS > 101 College St., Rm. 4-308 > Toronto, Ontario M5G 1L7 > > e: [EMAIL PROTECTED] > w: http://xtal.uhnres.utoronto.ca/prive > p: (416) 581-8550 ext. 7543 Sincerely yours, Mark A. White, Ph.D. Assistant Professor, Dept. Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Fax. (409) 747-4745 mailto://[EMAIL PROTECTED] http://xray.utmb.edu http://xray.utmb.edu/~white
