<div dir="ltr"><div dir="ltr">Hi Casper,<div> <br></div></div><div class="gmail_quote"><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div lang="DA"><div class="gmail-m_7009740232792741308WordSection1"><p class="MsoNormal"><span lang="EN-US">I am doing real space refinement on a protein model build from a cryo-em map. I run the refinement with Amber gradients turned on and with secondary structure restraints (otherwise, more or less default settings).</span></p></div></div></blockquote><div dir="ltr"><div>Amber gradients should not be relevant to the issue. SS filtration is based on input model geometry before any coordinate refinement was done.</div></div><div class="gmail_quote"><div></div></div><div> </div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div lang="DA"><div class="gmail-m_7009740232792741308WordSection1"><p class="MsoNormal"><span lang="EN-US"><u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">I have some issues with helix and sheet outliers.<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">Every time I run the refinement (after making sure that the bond distance in the helices are within the 3.5 Å) </span></p></div></div></blockquote><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div lang="DA"><div class="gmail-m_7009740232792741308WordSection1"><p class="MsoNormal"><span lang="EN-US">I get a lot of bad annotation remarks, but with outliers just above 3.5 Å (see .log below):<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">…<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New"">removed outlier: 3.576A pdb=" N VAL A 179 " --> pdb=" O LEU A 175 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> Proline residue: A 180 - end of helix<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 3.681A pdb=" N PHE A 188 " --> pdb=" O LEU A 184 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 3.628A pdb=" N PHE A 189 " --> pdb=" O ILE A 185 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 3.521A pdb=" N ILE A 190 " --> pdb=" O ALA A 186 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> Processing helix chain 'A' and resid 219 through 228<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 3.737A pdb=" N MET A 227 " --> pdb=" O GLU A 223 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">…<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">Is this something that I just need to fix in several rounds of refinement or is there an explanation?</span></p></div></div></blockquote><div>If you believe the distances you see in the log are wrong, I'm happy to investigate. Please send me the model file (off-list) with an explanation why do you think so. The distances (even 3.5) suggest a rather poor helix geometry since the target distance is 2.9A. If you are sure your annotations are correct, you may try to relax the threshold by setting parameter "distance_cut_n_o" to some larger value than default 3.5. Then the restraints will be imposed and hopefully the geometry of the helix will become good during refinement. </div><div> </div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div lang="DA"><div class="gmail-m_7009740232792741308WordSection1"><p class="MsoNormal"><span lang="EN-US"><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">In addition, I have some bad sheet annotations such as:<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">…<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New"">removed outlier: 6.987A pdb=" N VAL B 433 " --> pdb=" O LEU B 440 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 4.659A pdb=" N VAL B 442 " --> pdb=" O LEU B 431 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 6.296A pdb=" N LEU B 431 " --> pdb=" O VAL B 442 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 5.062A pdb=" N ALA B 444 " --> pdb=" O THR B 429 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US" style="font-family:"Courier New""> removed outlier: 6.129A pdb=" N THR B 429 " --> pdb=" O ALA B 444 " (cutoff:3.500A)<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">…<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">When I look at the model, the residues are definitely a part of the sheet – the long distance between the N-O atoms is due to the O being “flipped” (hope it makes sense).<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">Is this because of wrong annotation?</span></p></div></div></blockquote><div>If you believe the residues are part of the sheet - then the annotation is correct, the model is wrong. If you believe the O needs to be "flipped" - go ahead and flip it. Do any other adjustments you deem necessary to improve the model and hydrogen-bonding pattern. This may result in proper distance between N and O and established restraint. Such big model changes are better done manually rather than during refinement with restraints.</div><div><br></div><div>Some relevant tutorials are:</div><div><a href="https://www.youtube.com/watch?v=9dCkAdR1RDk&feature=youtu.be">https://www.youtube.com/watch?v=9dCkAdR1RDk&feature=youtu.be</a><br></div><div><a href="https://www.youtube.com/watch?v=qB8W_6yuw5k&feature=youtu.be">https://www.youtube.com/watch?v=qB8W_6yuw5k&feature=youtu.be</a><br></div><div><br></div><div>Relevant documentation and links in it:</div><div><a href="https://www.phenix-online.org/documentation/reference/secondary_structure.html">https://www.phenix-online.org/documentation/reference/secondary_structure.html</a><br></div><div><br></div><div>Please let us know if you have more questions.</div><div><br></div><div>Best regards,</div><div>Oleg Sobolev.</div><div> </div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div lang="DA"><div class="gmail-m_7009740232792741308WordSection1"><p class="MsoNormal"><span lang="EN-US"><u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US"><u></u> <u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">Best<u></u><u></u></span></p>
<p class="MsoNormal"><span lang="EN-US">Casper<u></u><u></u></span></p>
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