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Hi all,<br>
<br>
I am refining a structure that diffracted to 3.5 Angstroms and
contains 6 monomers in the ASU. It is noteworthy to mention, in
certain monomers, half their residues are missing (in other words no
density). Initial b-factors after molrep were 118.585, R=0.32, and
Rfree=0.40. Initial refinement was done with refmac and:<br>
<br>
<tt>mean b-value=117.344</tt><tt><br>
</tt><tt>R=0.31</tt><tt><br>
</tt><tt>Rfree=0.40</tt><br>
<br>
Refinement with Phenix
(phenix-installer-dev-2054-intel-linux-2.6-x86_64-centos5) was
performed and the Ramachandran plot was bad:<br>
<br>
<tt>OUTLIERS : 6.52 %</tt><tt><br>
</tt><tt>ALLOWED : 13.67 %</tt><tt><br>
</tt><tt>FAVORED : 79.82 %</tt><tt><br>
</tt><tt>ROTAMER OUTLIERS : 7.18 %</tt><tt><br>
</tt><tt>CBETA DEVIATIONS : 8</tt><br>
<br>
And <tt>final R=0.38</tt> and <tt></tt><tt>Rfree=0.41</tt>.<br>
<br>
I then improved Ramachandran plot in coot and follow Pavel's
suggestion:<br>
<br>
<meta http-equiv="content-type" content="text/html; charset=utf-8">
<pre style="color: rgb(0, 0, 0); font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; widows: 1; word-spacing: 0px; -webkit-text-stroke-width: 0px;"><i> - add H atoms:
</i>>><i>
</i>>><i> phenix.reduce model.pdb > model_H.pdb
</i>>><i>
</i>>><i> - then run a quick geometry regularization: <b>(</b><b>T</b><b>his part I </b><b>did in coot. phenix.</b><b>pdb</b><b>tools model_H.pdb --geom and phenix.pdbt</b><b>ools model_H.pdb --geometry</b><b>-</b><b>restraints do not work)</b>
</i>>><i>
</i>>><i> phenix.pdbtools model_H.pdb --geom
</i>>><i>
</i>>><i> - then strip off H:
</i>>><i>
</i>>><i> phenix.reduce -trim model_H_regularized.pdb > start.pdb
</i>>><i>
</i>>><i> - then run refinement using NCS and Ramachandran plot restraints:
</i>>><i>
</i>>><i> phenix.refine start.pdb def_data.mtz main.ncs=true --overwrite
</i>>><i> output.prefix=def2 main.number_of_mac=5 ramachandran_restraints=true
</i>>><i> rama_potential=emsley strategy=individual_sites+**individual_adp
</i>>><i>
</i>>><i> - then take outcome of previous refinement and run the same refinement
</i>>><i> plus weights optimization:
</i>>><i>
</i>>><i> phenix.refine def2_001.pdb def_data.mtz main.ncs=true --overwrite
</i>>><i> output.prefix=def3 main.number_of_mac=5 ramachandran_restraints=true
</i>>><i> rama_potential=emsley strategy=individual_sites+**individual_adp
</i>>><i> optimize_xyz=true nproc=6
</i><font face="sans-serif">My current stats are as follows:
</font><tt>Final R=0.35</tt><tt>
</tt><tt>Final Rfree=0.38</tt><tt>
</tt><tt>ALL-ATOM CLASHSCORE : 173.74</tt><tt>
</tt><tt>RAMACHANDRAN PLOT:</tt><tt>
</tt><tt>OUTLIERS : 2.27 %</tt><tt>
</tt><tt>ALLOWED : 4.61 %</tt><tt>
</tt><tt>FAVORED : 93.13 %</tt><tt>
</tt><tt>ROTAMER OUTLIERS : 36.26 %</tt><tt>
</tt><tt>CBETA DEVIATIONS : 6</tt><tt>
</tt><tt>Wilson B-Value: 108.4</tt><tt>
</tt><tt>Mean B-value: 108.997</tt><font face="sans-serif">
I am unsure how to proceed from here. I am planning on removing some side chains which have no density and then refining with Rama. restraints.
I can also send my mtz and pdb if that would help.
The stats when I processed the data are as follows from xia2 -3d (xds, xscale):
</font><tt>High resolution limit          3.50         15.65         3.50</tt><tt>
</tt><tt>Low resolution limit         109.96        109.96         3.59</tt><tt>
</tt><tt>Completeness          94.1         90.5         91.5</tt><tt>
</tt><tt>Multiplicity          4.8         5.1         3.2</tt><tt>
</tt><tt>I/sigma          11.3         26.5         2.4</tt><tt>
</tt><tt>Rmerge         0.118        0.068        0.458</tt><tt>
</tt><tt>Rmeas(I)         0.144        0.080        0.605</tt><tt>
</tt><tt>Rmeas(I+/-)         0.144        0.083        0.584</tt><tt>
</tt><tt>Rpim(I)         0.062        0.036        0.311</tt><tt>
</tt><tt>Rpim(I+/-)         0.081        0.047        0.358</tt><tt>
</tt><tt>CC half         0.992        0.994        0.791</tt><tt>
</tt><tt>Wilson B factor         90.052</tt><tt>
</tt><tt>Anomalous completeness          86.9         93.6         71.3</tt><tt>
</tt><tt>Anomalous multiplicity          2.4         3.0         1.8</tt><tt>
</tt><tt>Anomalous correlation         -0.262        -0.440        -0.052</tt><tt>
</tt><tt>Anomalous slope         1.005        0.000        0.000</tt><tt>
</tt><tt>Total observations         169836        2234        7765</tt><tt>
</tt><tt>Total unique         35071        442        2464</tt><tt>
</tt><tt>Assuming spacegroup: P 21 21 21</tt><tt>
</tt><tt>Unit cell:</tt><tt>
</tt><tt>73.010 180.780 219.910</tt><tt>
</tt><tt>90.000 90.000 90.000</tt><font face="sans-serif">
</font></pre>
Any help would greatly be appreciated,<br>
<pre class="moz-signature" cols="72">--
Edwin Lazo
Scientific Associate
Life Science and Biomedical Technology Research Resource
National Synchrotron Light Source II Building 745
Upton, NY 11973
Office:(631)344-7247
Fax:(631)344-5059</pre>
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