[phenixbb] phenix.refine Ile peptide bond breaks

Pavel Afonine PAfonine at lbl.gov
Wed May 6 20:57:06 PDT 2009

Hi Katherine,

sorry for the problem! From what you describe I can't really see what is 
wrong... So let's approach it this way:

- Are you using the latest version of PHENIX (or at least not older than 
a few months)? If not, please get the latest:
and try the refinement again.

- It is nearly impossible to tell what is wrong without actually looking 
at the PDB file. Could you please send me the PDB file? Also, I will 
need the exact command and parameters file (if you use one) that you use 
to run refinement. At this point I don't need the data, since I can 
simulate it given the PDB file. Please indicate the bonds that get broken.
I will look into this problem once I get the file and the command.


On 5/6/09 6:07 PM, SIPPEL,KATHERINE H wrote:
> 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
> --
> Ph. D. candidate
> McKenna Lab
> Department of Biochemistry and Molecular Biology
> College of Medicine
> University of Florida
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