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: [log in to unmask]
w: http://xtal.uhnres.utoronto.ca/prive
p: (416) 581-8550 ext. 7543
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