<div dir="ltr">Zixian Li<div><br></div><div>Some things that immediately require comment.</div><div><br></div><div>1. Coot is a separate tool from Phenix and it therefore does not know the things that phenix.refine knows. It does not use the automatic linking or restraints that Phenix uses. You need to supply the restraints that you want Coot to use. Unfortunately, it does not read the params files that Phenix uses. It relies on its internal coding to decide what is linked and what is not. Also <a href="http://phenix-online.org/newsletter/CCN_2016_01.pdf#page=10">http://phenix-online.org/newsletter/CCN_2016_01.pdf#page=10</a></div><div><br></div><div>2. Using the same data_link name "p" is not necessary so I would avoid it.</div><div><br></div><div>3. The only surefire way to know if a link has occurred in the refinement is to look in the .geo file. You may have some info in the log file also.</div><div><br></div><div>Having said that, I'm happy to look into the problem if it persists and you send me the files.</div><div><br clear="all"><div><div dir="ltr" class="gmail_signature" data-smartmail="gmail_signature"><div dir="ltr"><div><div dir="ltr"><div dir="ltr">Cheers<div><br></div><div>Nigel<div><br></div><div>---</div><div>Nigel W. Moriarty<br>Building 33R0349, Molecular Biophysics and Integrated Bioimaging</div><div>Lawrence Berkeley National Laboratory<br>Berkeley, CA 94720-8235<br>Phone : 510-486-5709 Email : NWMoriarty@LBL.gov<br>Fax : 510-486-5909 Web : <a href="http://CCI.LBL.gov" target="_blank">CCI.LBL.gov</a></div></div></div></div></div></div></div></div><br></div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Tue, Jul 7, 2020 at 1:09 PM Zixian Li <<a href="mailto:zixian.li@mail.mcgill.ca">zixian.li@mail.mcgill.ca</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">
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Dear Phenix Community,</div>
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I'm at beginner level of using Phenix. I have difficulty to refine a protein-RNA complex in Phenix.</div>
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I refined the protein model without RNA first, then I built the RNA model into the difference density map in Coot through Calculate>Other Modelling Tools>Ideal DNA/RNA, where I input the sequence and fit nucleotides into the density. At the 5' end it's guanosine
triphosphate (GTP) but the modeler tool in Coot only generates nucleoside monophosphate. <span style="margin:0px">So I built the RNA model from the second nucleotide at 5' end downstream until where RNA density was too poor to model. I did the real space
refine zone in Coot to fit this main part of the RNA into its density the best possibly.</span></div>
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For the GTP at 5' end, I realized there is GTP.cif restraint file in Phenix monomer library. So I obtained an arbitrary pdb file for GTP using eLBOW and corrected atom names matching the definition in GTP.cif file. Then I can also fit GTP into the RNA density
at 5' end using <span style="margin:0px;background-color:rgb(255,255,255);display:inline">real space refine zone in Coot. Now I have a broken RNA model with GTP unlinked to the <span style="margin:0px;background-color:rgb(255,255,255);display:inline">ribose-</span>phosphate
backbone. </span></div>
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For Phenix refinement, I supplied a cif_link_GTP-G.params file to define the linkage for the two RNA chains in the complex:</div>
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<span style="margin:0px">refinement.pdb_interpretation {<br>
</span>
<div style="margin:0px"> apply_cif_link {<br>
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<div style="margin:0px"> data_link = p<br>
</div>
<div style="margin:0px"> residue_selection_1 = chain C and resname GTP and resid 1<br>
</div>
<div style="margin:0px"> residue_selection_2 = chain C and resname G and resid 2<br>
</div>
<div style="margin:0px"> }<br>
</div>
<div style="margin:0px"> apply_cif_link {<br>
</div>
<div style="margin:0px"> data_link = p<br>
</div>
<div style="margin:0px"> residue_selection_1 = chain D and resname GTP and resid 1<br>
</div>
<div style="margin:0px"> residue_selection_2 = chain D and resname G and resid 2<br>
</div>
<div style="margin:0px"> }<br>
</div>
<span style="margin:0px">}</span><br>
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<span style="margin:0px">After refinement, I noticed O3' of GTP appears connected to P of the following nucleotide G in the refined model in Coot. However, if I do the real space refine zone in Coot, the linkage becomes broken, i.e., I cannot refine the RNA
model as a whole: Coot only allows separate real space refine for GTP and the rest. It seems Coot still cannot recognize GTP and the rest as one body. In the refinement settings there is a button for Automatic linking options.<span> </span><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px"> </span>Interestingly,<span style="margin:0px"> </span></span>I
noticed if I leave the automatic covalent linking as default, Phenix writes Link records in the output pdb after refinement. But it also links one of the protein residues to the GTP in chain D. In order to avoid the unwanted intermolecular linkage, I clicked
Link none for<span> </span><span style="margin:0px;background-color:rgb(255,255,255);display:inline">Automatic linking options</span>, then there is no any link records written in the output pdb despite of the defined linkage in <span style="margin:0px;background-color:rgb(255,255,255);display:inline">cif_link_GTP-G.params
file. So it seems for me Phenix ignores the input <span style="margin:0px;background-color:rgb(255,255,255);display:inline">cif_link_GTP-G.params file, it writes Link records only if the Automatic linking option is activated. I worried whether
it could be due to the syntax of the <span style="margin:0px;background-color:rgb(255,255,255);display:inline">cif_link_GTP-G.params file. So I tried the following syntax in two separate files, the same issue was observed.</span></span></span></span></div>
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<span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><br>
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<span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline">cif_link_GTP-G_chC.params<br>
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<span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px">refinement.pdb_interpretation.apply_cif_link
{<br>
</span>
<div style="margin:0px"> data_link = p<br>
</div>
<div style="margin:0px"> residue_selection_1 = chain C and resname GTP and resid 1<br>
</div>
<div style="margin:0px"> residue_selection_2 = chain C and resname G and resid 2<br>
</div>
<span style="margin:0px">}</span><br>
</span></span></span></span></div>
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<span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px"><br>
</span></span></span></span></span></div>
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<span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px">
<div style="margin:0px"><span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline">cif_link_GTP-G_chD.params<br>
</span></span></span></span></div>
<div style="margin:0px"><span style="margin:0px"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px;background-color:rgb(255,255,255);display:inline"><span style="margin:0px">refinement.pdb_interpretation.apply_cif_link
{<br>
</span>
<div style="margin:0px"> data_link = p<br>
</div>
<div style="margin:0px"> residue_selection_1 = chain D and resname GTP and resid 1<br>
</div>
<div style="margin:0px"> residue_selection_2 = chain D and resname G and resid 2<br>
</div>
<span style="margin:0px">}</span></span></span></span></span></div>
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I also need to point out that p linkage is pre-defined in the list of links (mon_lib_list.cif) in Phenix monomer library.<br>
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I apologize for this long explanation of the problem. I hope it is clear.</div>
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I really appreciate your advice in advance.</div>
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Sincerely,</div>
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Zixian Li</div>
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