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Hi Guillaume,</div>
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My RealSpaceRefinement starting structure (the output of NAMD MDFF refinement) is OK with respect to the D-DNA structure. It is not surprising as NAMD MDFF used SS restraints for DNA. So the B-DNA structure is actually distorted by RSR with phenix. </div>
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To test the impact of occupancy refinement, i set it the occupancy to 0.2 of the three 3' and 5' base pairs by editing the pdb file in input. No success there, the NA SS restraints does not permit to preserve the B-DNA structure in input. So i continue to investigate
this problem. </div>
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Best.</div>
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Philippe.</div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>De :</b> Guillaume Gaullier <guillaume.gaullier@kemi.uu.se><br>
<b>Envoyé :</b> mercredi 31 janvier 2024 10:42:43<br>
<b>À :</b> CUNIASSE Philippe<br>
<b>Cc :</b> Oleg Sobolev; phenixbb@phenix-online.org<br>
<b>Objet :</b> Re: [phenixbb] RealSpaceRefine Nucleic acid SS restraints ?</font>
<div> </div>
</div>
<div>Hello Philippe,
<div class=""><br class="">
</div>
<div class="">Regarding occupancy refinement, by default phenix.refine only refines atoms that already had a partial occupancy in the input model. This is explained here: <a href="https://phenix-online.org/documentation/reference/refinement.html#occupancy-refinement" class="">https://phenix-online.org/documentation/reference/refinement.html#occupancy-refinement</a></div>
<div class="">I don’t know if phenix.real_space_refine has the same default setting (you could find out by having it write out its defaults, I think there is a command-line option for this). But most likely, occupancy refinement is not the culprit if these
atoms had full occupancy in your starting model.</div>
<div class=""><br class="">
</div>
<div class="">Occupancy refinement does not seem warranted in this particular case anyway, since you said you don’t see clear alternative conformations but rather a very blurry density at the DNA ends. B-factor refinement would be better suited.</div>
<div class=""><br class="">
</div>
<div class="">Last, I remember that Tristan Croll noted somewhere (can’t remember if it was on this list or the chimerax-users list; maybe it’s also somewhere in ISOLDE’s documentation) that models from MD simulations systematically deviate from the bond length
and bond angle distributions that refinement programs target. I don’t remember the details, but it seems relevant to what you are doing so I figured I would point it out.</div>
<div class=""><br class="">
</div>
<div class="">I don’t know how to make the SS restraints apply correctly, but I hope these observations still help a bit!</div>
<div class="">Cheers,</div>
<div class="">
<div class="">
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<br class="Apple-interchange-newline">
Guillaume</div>
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<blockquote type="cite" class="">
<div class="">On 31 Jan 2024, at 07:49, CUNIASSE Philippe <<a href="mailto:Philippe.CUNIASSE@cea.fr" class="">Philippe.CUNIASSE@cea.fr</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
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<div style="margin-top: 0px; margin-bottom: 0px;" class="">Dear Oleg,</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">sorry for the log.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">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.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">I also checked the restraints in the .geo file and it seems correct. I only found in previ<span style="font-size: 12pt;" class="">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></div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">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.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Your advice on this hypothesis is welcome.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Best regards.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Philippe.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class=""><br class="">
</div>
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<div id="divRplyFwdMsg" dir="ltr" class=""><font face="Calibri, sans-serif" style="font-size: 11pt;" class=""><b class="">De :</b><span class="Apple-converted-space"> </span>Oleg Sobolev <<a href="mailto:osobolev@lbl.gov" class="">osobolev@lbl.gov</a>><br class="">
<b class="">Envoyé :</b><span class="Apple-converted-space"> </span>mardi 30 janvier 2024 18:21:02<br class="">
<b class="">À :</b><span class="Apple-converted-space"> </span>CUNIASSE Philippe<br class="">
<b class="">Cc :</b><span class="Apple-converted-space"> </span><a href="mailto:phenixbb@phenix-online.org" class="">phenixbb@phenix-online.org</a><br class="">
<b class="">Objet :</b><span class="Apple-converted-space"> </span>Re: [phenixbb] RealSpaceRefine Nucleic acid SS restraints ?</font>
<div class=""> </div>
</div>
<div class="">
<div dir="ltr" class="">Dear Philippe,
<div class=""><br class="">
</div>
<div class="">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 class=""><br class="">
</div>
<div class="">Couple of ways to check for this are: </div>
<div class="">1. Look in the .log into </div>
<div class=""> Finding SS restraints...<br class="">
Secondary structure from input PDB file:<br class="">
0 helices and 0 sheets defined<br class="">
0.0% alpha, 0.0% beta<br class="">
12 base pairs and 21 stacking pairs defined.<br class="">
Time for finding SS restraints: 0.04<br class="">
Creating SS restraints...<br class="">
<br class="">
No hydrogen bonds defined for protein.<br class="">
Restraints generated for nucleic acids:<br class="">
32 hydrogen bonds<br class="">
64 hydrogen bond angles<br class="">
0 basepair planarities<br class="">
12 basepair parallelities<br class="">
21 stacking parallelities<br class="">
</div>
<div class=""><br class="">
</div>
<div class="">This part would give a general idea if anything worked at all.</div>
<div class=""><br class="">
</div>
<div class="">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 class=""><br class="">
</div>
<div class="">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 class=""><br class="">
</div>
<div class="">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 class=""><br class="">
</div>
<div class="">Best regards,</div>
<div class="">Oleg Sobolev.</div>
</div>
<br class="">
<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" class="">Philippe.CUNIASSE@cea.fr</a>> wrote:<br class="">
</div>
<blockquote class="gmail_quote" style="margin: 0px 0px 0px 0.8ex; border-left-width: 1px; border-left-style: solid; border-left-color: rgb(204, 204, 204); padding-left: 1ex;">
<div class="msg-2061162240245066178">
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<div style="margin-top: 0px; margin-bottom: 0px;" class="">Dear all,</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">I am currently refining a protein-nucleic acid structure obtained by MDFF (NAMD) with a 2.9 Å cryo-EM map.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">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.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">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;" class="">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></div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Have you a suggestion to solve this problem ?</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Thanks in advance for your help.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Best regards.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class="">Philippe.</div>
<div style="margin-top: 0px; margin-bottom: 0px;" class=""><br class="">
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<span lang="EN-US" style="font-family: Courier;" class="">Philippe Cuniasse, PhD/HDR.<u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier; color: rgb(51, 51, 51);" class="">Institute for Integrative Biology of the Cell (I2BC)</span><span lang="EN-US" style="font-family: Courier;" class=""><u class=""></u><u class=""></u></span></div>
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<span style="font-family: Courier;" class="">UMR 9198 CNRS-CEA-Univ Paris Sud<u class=""></u><u class=""></u></span></div>
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<span style="font-family: Courier;" class="">Bat 144 CE-Saclay<u class=""></u><u class=""></u></span></div>
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<span style="font-family: Courier;" class="">91191 Gif-sur-Yvette Cedex, France<u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier;" class="">Tel: (33) 1 69 08 56 35<u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier;" class="">Fax: (33) 1 69 08 47 12<u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier;" class="">Email: </span><span style="font-family: Courier; color: rgb(0, 0, 254);" class=""><a href="mailto:philippe.cuniasse@cea.fr" title="mailto:philippe.cuniasse@cea.fr" target="_blank" style="color: rgb(5, 99, 193);" class=""><span lang="EN-US" style="color: purple;" class="">philippe.cuniasse@cea.fr</span></a></span><span lang="EN-US" style="font-family: Courier;" class=""><u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier;" class="">Web:</span><span lang="EN-US" style="font-family: Courier; color: rgb(0, 0, 245);" class=""> </span><span style="font-family: Courier; color: rgb(0, 0, 245);" class=""><a href="http://biodev.cea.fr/rasmot3d/" title="http://biodev.cea.fr/rasmot3d/" target="_blank" style="color: rgb(5, 99, 193);" class=""><span lang="EN-US" style="color: purple;" class="">http://biodev.cea.fr/rasmot3d/</span></a></span><span lang="EN-US" style="font-family: Courier; color: rgb(0, 0, 245);" class=""><u class=""></u><u class=""></u></span></div>
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<span lang="EN-US" style="font-family: Courier; color: rgb(0, 0, 245);" class="">Web:<span class=""> </span><a href="https://www.i2bc.paris-saclay.fr/" title="https://www.i2bc.paris-saclay.fr/" target="_blank" style="color: rgb(5, 99, 193);" class=""><span style="color: rgb(5, 99, 193);" class="">https://www.i2bc.paris-saclay.fr</span></a><u class=""></u><u class=""></u></span></div>
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