<div dir="ltr"><div dir="ltr">Hi Yangqi,<div><br></div><div>Assuming that your map has perfect helical symmetry, the information in your map is just the information for one monomer, plus information about contacts.</div><div><br></div><div>With that in mind, the only information refinement can provide you about most of your molecule is what you get when you refine just one molecule by itself. Refinement of the filament in addition will give you information about the contacts (by excluding bad contacts).</div><div><br></div><div>As you note, at 3.8 A you don't have clear details about everything in the map.</div><div><br></div><div>So...I suggest refining the monomer carefully as you have. Then as you did, take the fixed monomer, make a filament with it, and refine with rigid-body refinement. </div><div><br></div><div>Next, have a look at the contacts between monomers. By hand, or by refinement of just 2 monomers, fix everything at the contacts in one monomer. </div><div><br></div><div>Now make a new filament with the fixed monomer, refine rigid body again...etc until you have all good contacts and a filament.</div><div><br></div><div>You might try more with finding helical symmetry in your map as well. You can generate regular helical symmetry automatically with map_symmetry, specifying the operators. That could help as well.</div><div><br></div><div>All the best,</div><div>Tom T</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Tue, Jan 21, 2020 at 7:32 AM BuddySphinx <<a href="mailto:yangqi.gu@yale.edu">yangqi.gu@yale.edu</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"><div lang="EN-US"><div class="gmail-m_-2531535597394312784WordSection1"><p class="MsoNormal">Hi Tom,</p><p class="MsoNormal">Thank you for your detailed answers. I did refinement with or without NCS restraints (But in both cases, I did not provide any NCS operator. So I assume what it does is C1 symmetry?). Without NCS restraints refinement always outperform with the NCS restraint, such as Ramachandra, bond, angle, rotamer outliers. However, the biggest issue I have right now is that after refinement, not every chain is identical since they were refined independently? From my understanding, at the resolution I have (~3.8 A), it is better to constrain them to be identical? Am I correct? I refined the monomer first and then I built the model based on refined monomer. Also, for the torsion angle, do I have to specify from the command line or it is already implemented in gui interface?</p><p class="MsoNormal">Best,</p><p class="MsoNormal">Yangqi</p><p class="MsoNormal"><u></u> <u></u></p><p class="MsoNormal">Sent from <a href="https://go.microsoft.com/fwlink/?LinkId=550986" target="_blank">Mail</a> for Windows 10</p><p class="MsoNormal"><u></u> <u></u></p><div style="border-right:none;border-bottom:none;border-left:none;border-top:1pt solid rgb(225,225,225);padding:3pt 0in 0in"><p class="MsoNormal" style="border:none;padding:0in"><b>From: </b><a href="mailto:tterwilliger@newmexicoconsortium.org" target="_blank">Tom Terwilliger</a><br><b>Sent: </b>Tuesday, January 21, 2020 9:18 AM<br><b>To: </b><a href="mailto:yangqi.gu@yale.edu" target="_blank">Yangqi Gu</a>; <a href="mailto:tterwilliger@newmexicoconsortium.org" target="_blank">Thomas Charles Terwilliger</a><br><b>Cc: </b><a href="mailto:phenixbb@phenix-online.org" target="_blank">PHENIX user mailing list</a><br><b>Subject: </b>Re: [phenixbb] How critical is ncs operator for real space refinement</p></div><p class="MsoNormal"><u></u> <u></u></p><div><div><p class="MsoNormal">Hi Yangqi,</p></div><div><p class="MsoNormal"><u></u> <u></u></p><div><p class="MsoNormal">You don't actually need the NCS relationships in order to refine with NCS restraints because the default is to use torsion-angle restraints which only use local relationships, not overall NCS matrices.</p></div><div><p class="MsoNormal"><u></u> <u></u></p></div><div><p class="MsoNormal">So you can go ahead and use your rigid-body-refined starting model with torsion-angle restraints in phenix.real_space_refine. That will not impose perfect helical restraints, but I am guessing that your map already has near-perfect helical symmetry so that should not matter very much.</p></div><div><p class="MsoNormal"><u></u> <u></u></p></div><div><p class="MsoNormal">Also you might first run with just one monomer which will be much quicker and give you almost the same answer.</p></div><div><p class="MsoNormal"><u></u> <u></u></p></div><div><p class="MsoNormal">Let us know if that does not do it!</p></div><div><p class="MsoNormal"><u></u> <u></u></p></div><div><p class="MsoNormal">All the best,</p></div><div><p class="MsoNormal">Tom T</p></div><div><p class="MsoNormal"><u></u> <u></u></p></div></div><p class="MsoNormal"><u></u> <u></u></p><div><div><p class="MsoNormal">On Mon, Jan 20, 2020 at 9:33 PM Yangqi Gu <<a href="mailto:yangqi.gu@yale.edu" target="_blank">yangqi.gu@yale.edu</a>> wrote:</p></div><blockquote style="border-top:none;border-right:none;border-bottom:none;border-left:1pt solid rgb(204,204,204);padding:0in 0in 0in 6pt;margin-left:4.8pt;margin-right:0in"><div><p class="MsoNormal">Dear PHENIX developers,</p><div><p class="MsoNormal">I have a question about NCS operator. Since I have a cryo-EM map and I want to refine my filament coordinates within the map. I generated the coordinate map manually in Chimera by building multiple chains with rigid body refinement. Since I did not generate the NCS operator file (I tried by doing map symmetry, it did not give me the right helical symmetry), I did not refine with ncs restraints. I am wondering in this case, how critical it is to refine a model with helical symmetry applied. Is it a must or can I go with the way I did? Hope to hear from you soon!</p></div><div><p class="MsoNormal">Best,</p></div><div><p class="MsoNormal">Yangqi<br clear="all"></p><div><p class="MsoNormal"><u></u> <u></u></p></div></div></div><p class="MsoNormal">_______________________________________________<br>phenixbb mailing list<br><a href="mailto:phenixbb@phenix-online.org" target="_blank">phenixbb@phenix-online.org</a><br><a href="http://phenix-online.org/mailman/listinfo/phenixbb" target="_blank">http://phenix-online.org/mailman/listinfo/phenixbb</a><br>Unsubscribe: <a href="mailto:phenixbb-leave@phenix-online.org" target="_blank">phenixbb-leave@phenix-online.org</a></p></blockquote></div><p class="MsoNormal"><br clear="all"></p><div><p class="MsoNormal"><u></u> <u></u></p></div><p class="MsoNormal">-- </p><div><div><div><div><div><div><div><div><div><p class="MsoNormal">Thomas C Terwilliger</p><div><p class="MsoNormal">Laboratory Fellow, Los Alamos National Laboratory</p></div><div><p class="MsoNormal">Senior Scientist, New Mexico Consortium</p></div><div><p class="MsoNormal">100 Entrada Dr, Los Alamos, NM 87544</p></div><div><p class="MsoNormal">Email: <a href="mailto:tterwilliger@newmexicoconsortium.org" target="_blank">tterwilliger@newmexicoconsortium.org</a></p></div><div><p class="MsoNormal">Tel: 505-431-0010</p></div></div></div></div></div></div></div></div></div></div></div><p class="MsoNormal"><u></u> <u></u></p><p class="MsoNormal"><u></u> <u></u></p></div></div>
</blockquote></div><br clear="all"><div><br></div>-- <br><div dir="ltr" class="gmail_signature"><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div><div dir="ltr"><div dir="ltr">Thomas C Terwilliger<div>Laboratory Fellow, Los Alamos National Laboratory</div><div>Senior Scientist, New Mexico Consortium</div><div>100 Entrada Dr, Los Alamos, NM 87544</div><div>Email: <a href="mailto:tterwilliger@newmexicoconsortium.org" target="_blank">tterwilliger@newmexicoconsortium.org</a></div><div>Tel: 505-431-0010</div><div><br></div></div></div></div></div></div></div></div></div></div></div>