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<p>Hi Philippe,</p>
<p><br>
occupancy refinement should not affect coordinate refinement at
all in any way.</p>
<p><br>
</p>
<p>What happens if you run pure geometry regularization using your
input model and SS restraints? In this case there is no
experimental data involved whatsoever and the resulting
regularized model *must* literally obey the geometry specified by
the restraints (with some possibly tiny deviations in case
regularization has not converged):</p>
<p><br>
</p>
<p>phenix.geometry_minimization model.pdb restraints.eff<br>
</p>
<p><br>
</p>
<p>where restraints.eff specify your SS restraints.</p>
<p><br>
</p>
<p>If in this case restraints are still not obeyed in the
regularized model, please send us input files and we will
investigate.</p>
<p><br>
Pavel</p>
<p><br>
</p>
<p><br>
</p>
<div class="moz-cite-prefix">On 1/30/24 22:49, CUNIASSE Philippe
wrote:<br>
</div>
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<p>Dear Oleg,</p>
<p>sorry for the log.</p>
<p>The strange thing is that the input structure (output from
NAMD MDFF where we also apply SS restraints to NA has a
correct and better SS than the one at the end of the
RealSpace refine.</p>
<p>I also checked the restraints in the .geo file and it seems
correct. I only found in previ<span style="font-size: 12pt;">ious
calculations that the atom identity of some atoms (in the
charmm 36 topology) must be changed for the NA restraints
to be correctly set by phenix (C3M of THY must be changed
for C7). </span></p>
<p>My only hypothesis at the moment is that the occupancy
refinement (currently at a value of 1.00 for all
atoms) tends to distort strongly the extremities of the
duplex because the electron density is weak in these regions
with a bad shape and that this (presumlably) distorts the NA
structure to fit the poor density that does not ressemble
the one for a B-DNA in these regions. This could be solved
if we have a way to increase the harmonic constants for the
SS restraints, but i dont see this possibility.</p>
<p>Your advice on this hypothesis is welcome.</p>
<p>Best regards.</p>
<p>Philippe.</p>
<p><br>
</p>
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style="color: rgb(33, 33, 33); font-family: wf_segoe-ui_normal, "Segoe UI", "Segoe WP", Tahoma, Arial, sans-serif, serif, EmojiFont; font-size: 13.3333px;">Philippe
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<div id="divRplyFwdMsg" dir="ltr"><font style="font-size:11pt"
face="Calibri, sans-serif" color="#000000"><b>De :</b> Oleg
Sobolev <a class="moz-txt-link-rfc2396E" href="mailto:osobolev@lbl.gov"><osobolev@lbl.gov></a><br>
<b>Envoyé :</b> mardi 30 janvier 2024 18:21:02<br>
<b>À :</b> CUNIASSE Philippe<br>
<b>Cc :</b> <a class="moz-txt-link-abbreviated" href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a><br>
<b>Objet :</b> Re: [phenixbb] RealSpaceRefine Nucleic acid
SS restraints ?</font>
<div> </div>
</div>
<div>
<div dir="ltr">Dear Philippe,
<div><br>
</div>
<div>Most often the problem is that the geometry of the
input model is so distorted that the procedure fails to
find base pairs and therefore restrain them. </div>
<div><br>
</div>
<div>Couple of ways to check for this are: </div>
<div>1. Look in the .log into </div>
<div> Finding SS restraints...<br>
Secondary structure from input PDB file:<br>
0 helices and 0 sheets defined<br>
0.0% alpha, 0.0% beta<br>
12 base pairs and 21 stacking pairs defined.<br>
Time for finding SS restraints: 0.04<br>
Creating SS restraints...<br>
<br>
No hydrogen bonds defined for protein.<br>
Restraints generated for nucleic acids:<br>
32 hydrogen bonds<br>
64 hydrogen bond angles<br>
0 basepair planarities<br>
12 basepair parallelities<br>
21 stacking parallelities<br>
</div>
<div><br>
</div>
<div>This part would give a general idea if anything worked
at all.</div>
<div><br>
</div>
<div>2. Inspect .geo file after refinement and look for
"Basepair parallelity restraints" and "Bond-like
restraints" (secondary structure H-bonds) sections. Here
you can see exactly what restraints were applied and if
the basepairs in question were in fact restrained.</div>
<div><br>
</div>
<div>If you supply the restraints in the .parameter file,
check the nucleic_acid.hbond_distance_cutoff parameter.
The default is 3.4, you may need to increase it so your
annotations are not filtered out.</div>
<div><br>
</div>
<div>I'm happy to look at your files (model and parameter,
if any) to figure out what is happening. Please send them
off-list indicating problematic residues. Files will be
treated confidentially.</div>
<div><br>
</div>
<div>Best regards,</div>
<div>Oleg Sobolev.</div>
</div>
<br>
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">On Tue, Jan 30, 2024 at
7:20 AM CUNIASSE Philippe <<a
href="mailto:Philippe.CUNIASSE@cea.fr"
moz-do-not-send="true" class="moz-txt-link-freetext">Philippe.CUNIASSE@cea.fr</a>>
wrote:<br>
</div>
<blockquote class="gmail_quote"
style="margin:0px 0px 0px 0.8ex; border-left:1px solid rgb(204,204,204); padding-left:1ex">
<div class="msg-2061162240245066178">
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<p>Dear all,</p>
<p>I am currently refining a protein-nucleic acid
structure obtained by MDFF (NAMD) with a 2.9 Å
cryo-EM map.</p>
<p>Dues to the poor density of the map at the end of
the DNA duplex (likely due to breathing at the
ends of the duplex), the structure of the DNA
tends to be distorted.</p>
<p>I tried to set up a set of nucleic acid
restraints (Base pairing and base stacking for the
full sequence), h<span style="font-size:12pt">owever,
despite the fact that the syntax was checked and
no error message is present in the log file, it
seems that the restraints are not taken into
account as if the constant restraints were two
weak. And indeed when examining the refined
structure, the pairing and stacking restraints
for the base pairs at the extremities of the DNA
duplex are not fulfilled.</span></p>
<p>Have you a suggestion to solve this problem ?</p>
<p>Thanks in advance for your help.</p>
<p>Best regards.</p>
<p>Philippe.</p>
<p><br>
</p>
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<span style="font-family:Courier" lang="EN-US">Philippe
Cuniasse, PhD/HDR.</span></p>
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