[phenixbb] High B Factors

[Miles Pufall]

Hello All - 

I am working on a kinase:inhibitor complex with some interesting properties.  First, it is a long, thin unit cell (45x45x300) that has highly anisotropic B-factors (60 in two dimensions and 35 in the other according to xtriage).  We had a fair amount of trouble collecting data for these complexes due to somewhat high mosaicity (1.5) and overlap trouble, mostly due to the long unit cell.  As a result, although we get diffraction out to ~2.3 A with good signal (I/sigma of 3), the completeness is only OK - dropping off from a high of 95% around 3.5 A pretty much linearly to 75% at 2.3.  

As a result, the maps look only OK for 2.3A data, but clearly show the interactions between the inhibitor and the kinase that we are looking to define.  In that sense, the experiment worked just fine.  That said, the structure looks pretty good, although about 1/3 shows poor density for sidechains, which I wouldn't expect at this resolution.  But as I put the finishing touches on the structure a couple of things have worried me - first, there is consistently a larger than average gap between R and Rfree (Typically in the R~22 and Rfree~29).  This, according to Polygon, is within the range of reported structures, so I think that's OK. What is odd is that despite having an average B caluclated from the reflection of ~50, the average B after refinement is ~80, which is very much on the high end of what has been reported.  I have played around a lot with the wxc/wxu balance, and although this can change the Rs and tends to tighten the geometry perhaps inappropriately (rms bond ~0.003, rms angle ~0.7), the B factors stay very high.  

My question is, should I worry about this?  And if so, what can you recommend trying.  As usual, I'm happy to provide data if it help elucidate the problem.

Thanks in advance -

M

Miles Pufall
Postdoctoral Scholar
Yamamoto Lab
UC San Francisco
Cellular and Molecular Pharmacology
Mail Stop 2280
600 16th Street, Genentech Hall S-574
San Francisco, California 94158-2517
(415)476-4480


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