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<p class="MsoNormal">Hello !<o:p></o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><span lang="EN-US">Here I go asking for help again… <o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">I’m finishing the refinement of a ~800 residues structure at 2.4 angstroms, the protein sample was extracted from an animal, it has a few PTMs, especially on lysine side chains. I’m used to placing M3L and running Buster
on them, no problems. But now with this new protein, when I place a dimethyl-lysine (MLY) as amino acid 189, something very strange happens (and I tried this twice already):<o:p></o:p></span></p>
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoListParagraph" style="margin-left:0cm;mso-list:l1 level1 lfo1"><span lang="EN-US">Buster places a TER line just upstream to the MLY residue<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l1 level1 lfo1"><span lang="EN-US">The PDB Validate server interprets everything downstream to aa 188 as HETATM, so all the peptide bonds are considered as clashes – funny enough, on Coot everything
looks perfectly fine, Rama and planarity of the two peptide bonds around the modified lysine are fine…<o:p></o:p></span></li></ol>
<p class="MsoNormal"><span lang="EN-US">My detailed protocol for adding modified residues like this one is:<o:p></o:p></span></p>
<ol style="margin-top:0cm" start="1" type="1">
<li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Delete the original LYS<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Coot: File->Get Monomer… MLY<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Place it in the density, do some real space refinement
<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Delete OXT, H1 and H2 (otherwise the peptide bond gets 3 hydrogens bound to the N).<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Edit->Merge Molecules… (this usually gives the right chain name to the residue already)<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Edit->Renumber Residues… to put it in the sequence<o:p></o:p></span></li><li class="MsoListParagraph" style="margin-left:0cm;mso-list:l0 level1 lfo2"><span lang="EN-US">Real space refinement again, to get the peptide bond correct.<o:p></o:p></span></li></ol>
<p class="MsoNormal"><span lang="EN-US">I am sure it is Buster, and not Coot, adding the TER line.
<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">I also tried producing new restrains for MLY with Grade and running Buster with
<i>AnalyseGellySanityCheckForDuplicateBonds=no</i> – but the TER line still appears<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Of course I can cheat and modify the final mmcif file so that Validate will probably accept it, but I imagine there is a way to tell Buster to behave just like with M3L? We have a few similar structures that will be published
in the same paper…<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">I hope you can help, because the other refinement programs always give worse geometry than Buster on these cases, I didn’t even try them.<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Best,<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="color:black;mso-ligatures:none;mso-fareast-language:FR">---------------------------------------------------<br>
Carlos KIKUTI, PhD<br>
UMR144 - CNRS - Institut Curie<o:p></o:p></span></p>
<p class="MsoNormal"><span style="color:black;mso-ligatures:none;mso-fareast-language:FR">Pavillon Trouillet Rossignol <br>
26 Rue d’Ulm - 75005 Paris, France<br>
<a href="mailto:carlos.kikuti@curie.fr" title="mailto:carlos.kikuti@curie.fr"><span style="color:#0563C1">carlos.kikuti@curie.fr</span></a><o:p></o:p></span></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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