I guess I would go back to the data.
Could there be twinning?
Is the a pseudo translation vector which means there are systematically
weak zones of data? ( hklview might show something strange)
Rfree seems rather close to R to me for this resolution - maybe it
should go up a bit?
How many cycles have you done already? PHaser refines without taking
FreeR into account I think..
And is there density for the missing bits?
Eleanor
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: [log in to unmask]
> w: http://xtal.uhnres.utoronto.ca/prive
> p: (416) 581-8550 ext. 7543
>
>
>
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