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
