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Hi Michael,<br>
<br>
can you send me the PDB file (please send it off-list)? It's hard to
tell anything without looking at the file.<br>
<br>
Pavel.<br>
<br>
On 5/13/11 5:23 AM, R.M. Garavito wrote:
<blockquote cite="mid:27F524B8-DCB7-4CBF-81D9-33DEB5E7137E@msu.edu"
type="cite">
<div>We are trying to finish up the refinement on two structures,
but we have run into a strange problem that I have never seen
before. So we need to ask the Phenix experts. As background,
we recently solved the structure of an 808 residue enzyme, which
crystallizes as a tetramer. In the asymmetric unit there are
two tetramers (>6400 residues, ~52,000 atoms, but 67%
solvent). After SAD phasing and autobuild (many kudos to the
Phenix team), we were able to trace easily the chain in two
different ligand complexes, one at 2.85 Å and the other at 2.8
Å. Although the C-terminal domains are quite well resolved (and
obey 222 NCS), the first domain (~110 residues out of 808) is a
bit disordered and shows some conformational differences between
each monomer. While the main chain in this domain can be
traced, side chain densities are mediocre to missing in spots.
In order to ensure a consistent tracing and sequence assignment
between all 8 N-terminal domain, we built in the "correct"
sequence, but truncated the side chains to CB if the side chain
density was poor to non-existent. After several rounds of
refinement and model building, the finished structures look
quite good, have a consistent sequence assignment, and have
Rwork in the high teens and Rfree in the low 20s.</div>
<div><br>
</div>
<div>THE PROBLEM: It is the 2.8 Å complex that has the problem.
We used Moleman2 to evaluate the B-factors for the 4 domains in
the 8 monomers in the ASU. When we compiled the numbers, we
noticed that Moleman2 and phenix.refine gave different numbers
for the protein atoms for chain B in the 2.8 Å complex (6152
versus 6289). We checked the PDB and confirmed that there are
6152 protein atoms in chain B; the other 7 chains show no
differences between the programs. So where did phenix.refine
get 6289 protein atoms when only 6152 protein atoms are input
for chain B? The number of residues are correct (780).</div>
<div><br>
</div>
<div>Adding to the confusion, there are truncated residues in each
of the 8 chains. In the phenix.refine output attached below,
the PDB file is being read in. For chain A, the number of atoms
and the number of residues are correct, as well as the number of
incomplete residues ({'truncation_to_alanine': 14}). However,
for chain B, the number of incomplete residues
({'truncation_to_alanine': 21}) should be 37.</div>
<div><br>
</div>
<div>Any ideas?</div>
<div><br>
</div>
<div>Thanks,</div>
<div><br>
</div>
<div>Michael</div>
<div><br>
</div>
<div apple-content-edited="true">
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<p style="margin: 0px;"><font style="font: 12px Courier;"
face="Courier" size="3"><i style="font-family: Courier;
font-size: 12px; font-style: italic;"><span
class="Apple-style-span" style="font-family: Courier;
font-size: 12px; font-style: italic;">R. Michael
Garavito, Ph.D.</span></i></font></p>
<p style="margin: 0px;"><font style="font: 12px Courier;"
face="Courier" size="3"><i style="font-family: Courier;
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& Molecular Biology</span></i></font></p>
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</span></span></i></font></p>
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<p style="margin: 0px;"><font style="font: 12px Courier;"
face="Courier" size="3"><i style="font-family: Courier;
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</div>
<span class="Apple-style-span" style="font-size: 14px;"><span
class="Apple-style-span" style="font-size: medium;">phenix.refine
output:</span></span></div>
<div apple-content-edited="true"><span class="Apple-style-span"
style="font-size: 14px;"><br>
</span></div>
<div> Monomer Library directory:</div>
<div> "/usr/local/phenix-1.7-650/chem_data/mon_lib"</div>
<div> Total number of atoms: 51463</div>
<div> Number of models: 1</div>
<div> Model: ""</div>
<div> Number of chains: 57</div>
<div> Chain: "A"</div>
<div> Number of atoms: 6289</div>
<div> Number of conformers: 1</div>
<div> Conformer: ""</div>
<div> Number of residues, atoms: 781, 6289 <====
matches PDB file</div>
<div> Classifications: {'peptide': 781}</div>
<div> Incomplete info: {'truncation_to_alanine': 14}
<==== MATCHES PDB file</div>
<div> Link IDs: {'PTRANS': 34, 'TRANS': 746}</div>
<div> Unresolved non-hydrogen bonds: 47</div>
<div> Unresolved non-hydrogen angles: 60</div>
<div> Unresolved non-hydrogen dihedrals: 36</div>
<div> Unresolved non-hydrogen chiralities: 7</div>
<div> Planarities with less than four sites: {'PHE:plan':
1, 'GLU:plan': 3, 'ASN:plan2': 1, 'ASN:plan1': 1}</div>
<div> Unresolved non-hydrogen planarities: 23</div>
<div> .</div>
<div> .</div>
<div> .</div>
<div> .</div>
<div> Chain: "B"</div>
<div> Number of atoms: 6236</div>
<div> Number of conformers: 1</div>
<div> Conformer: ""</div>
<div> Number of residues, atoms: 780, 6236 <==== DOES
NOT MATCH PDB file 780 residues with 6152 atoms</div>
<div> Classifications: {'peptide': 780}</div>
<div> Incomplete info: {'truncation_to_alanine': 21}
<==== 37 truncation_to_alanine</div>
<div> Link IDs: {'PTRANS': 34, 'TRANS': 745}</div>
<div> Unresolved chain links: 1</div>
<div> Chain breaks: 1</div>
<div> Unresolved chain link angles: 3</div>
<div> Unresolved chain link dihedrals: 4</div>
<div> Unresolved chain link planarities: 2</div>
<div> Unresolved non-hydrogen bonds: 80</div>
<div> Unresolved non-hydrogen angles: 101</div>
<div> Unresolved non-hydrogen dihedrals: 62</div>
<div> Unresolved non-hydrogen chiralities: 9</div>
<div> Planarities with less than four sites:
{'GLN:plan1': 1, 'GLN:plan2': 1, 'ASP:plan': 1, 'PHE:plan': 1,
'GLU:plan': 6, 'ARG:plan': 2}</div>
<div> Unresolved non-hydrogen planarities: 49</div>
<div><br>
</div>
<pre wrap="">
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