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<p class="MsoNormal"><span lang="EN-US">Hi..<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 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).<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">I have some issues with helix and sheet outliers.<o:p></o:p></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 �) I get a lot of bad annotation remarks, but with outliers just above 3.5 � (see .log below):<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">…<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><span lang="EN-US" style="font-family:"Courier New""> Proline residue: A 180 - end of helix<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><span lang="EN-US" style="font-family:"Courier New""> Processing helix chain 'A' and resid 219 through 228<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<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"><o:p> </o:p></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? Amber gradients maybe?<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">In addition, I have some bad sheet annotations such as:<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">…<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<o:p></o:p></span></p>
<p class="MsoNormal" style="text-autospace:none"><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)<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"><o:p> </o:p></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).<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US">Is this because of wrong annotation?<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"><o:p> </o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="mso-fareast-language:DA">Best<o:p></o:p></span></p>
<p class="MsoNormal"><span lang="EN-US" style="mso-fareast-language:DA">Casper<o:p></o:p></span></p>
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