[phenixbb] phenix.refine Ile peptide bond breaks
SIPPEL,KATHERINE H
ksippel at ufl.edu
Wed May 6 18:07:57 PDT 2009
Hi all,
I've got a 2.1 angstrom structure that I am refining. The space
group is P212121. The structure is a dimer which I am refining
without NCS restraints. Each monomer has 333 residues and one
ligand, there are 33 Ile residues in each monomer. The first
couple of rounds of refinement I had no problems, but during the
third round phenix started breaking the main chain at the peptide
bond and the side chain between CA and CB on some of the Ile
residues. Specifically there are two breaks in chain A and four
breaks in chain B, with two additional residues in chain B
breaking only the CA-CB bond. Only one of the breaks is between
the same residue in both chains.
I'll be boring and run through my refinement process. This is the
first time I've used phenix for anything but AutoSol and it is
very likely that I have messed something up. Feel free to correct
anything else I've done wrong along the way.
Round 1, I rigid body refined chain A and B independently,
performed simulated annealing, and refined individual coordinates.
In COOT I manually refined and removed some bad loops. A few of
the Ile were deleted but none of the problem ones
Round 2, I refined individual_sites and individual_adp and turned
off simulated annealing. In COOT I rebuilt some of the loops and
fitted the ligand into the density. I generated a .cif file in
eLBOW.
Round 3, I refined the same as round 2 except that I included the
.cif file. This is when I got the peptide bond breaks for Ile
residues. I figured it was a fluke, fixed the breaks in COOT and
rebuilt a few more residues in the loops.
I ran another round of refinement and the same breaks showed up in
the pdb file. I double checked the .geo file to see if Real Space
Refine had made the bond distances were too long, but all the
input distances were within one or two hundredths of an angstrom
from ideal.
I considered it was maybe the ADP refinement as I was borderline
pushing the number of parameters I was refining given the number
of reflections I had. I turned off the individual_adp refinement
and reran refine_003. No luck.
I tried increasing the weight of the geometry restraints over the
observed data. I did this by increasing target_weights.wxc_scale
= 1.5. (I suspect that I got this wrong. As I understood the
wxc_scale is the ratio of the geometry restraints to the observed
data. Feel free to point and laugh as long as you also provide an
explanation as to what this parameter really means and how I
should have done it,please.) Still no luck.
For future information none of the problem isoleucines were
located near the ligand or near any of the loops I was
rebuilding.
I think that's everything. Any help would be appreciated,
Thanks,
Kat
--
SIPPEL,KATHERINE H
Ph. D. candidate
McKenna Lab
Department of Biochemistry and Molecular Biology
College of Medicine
University of Florida
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