<div dir="ltr"><div>Thanks so much Pavel !</div><div><br></div><div>I already went through placing the crystal structures manually in the density map and building the entire model. However, I didn't use the reference model restrains during the refinement and that explains why every time I refine, the geometry goes bad. I'll do this as you suggested.</div><div><br></div><div>Best,</div><div>Ashu<br></div></div><br><div class="gmail_quote"><div dir="ltr">On Tue, Nov 6, 2018 at 3:47 AM Pavel Afonine <<a href="mailto:pafonine@lbl.gov">pafonine@lbl.gov</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">Hi Ashu,<br>
<br>
you can use phenix.dock_in_map to dock known pieces (from crystal <br>
structures) into cryo-EM map.<br>
<br>
Then you can use phenix.real_space_refine to refine placed model into <br>
cryo-EM map. It is important that you use reference model restraints <br>
with high-res X-ray model as a reference.<br>
<br>
What to do with side chains is a good question. I doubt you can see any <br>
side chains in 7A resolution map. Reference X-ray model is perhaps your <br>
best source of knowledge about the side chains. Though keeping them in <br>
the model in the absence of support from the data seems questionable. I <br>
doubt there is a clear cut consensus/answer to this.<br>
<br>
You can vary the strength of reference model restraints do vary the dose <br>
of bias from the known (reference) model.<br>
<br>
Pavel<br>
<br>
> Dear All,<br>
><br>
> I am refining a ~7A single particle cryo-em structure of a protein. <br>
> The protein has two domains and high-resolution crystal structures of <br>
> individual domains are already known. I could fit these two crystal <br>
> structures in the low resolution cryoem map of the full-length <br>
> protein. I have two questions:<br>
><br>
> 1) Is it acceptable to refine with side chains in this case? In most <br>
> of the cases, with the guidane from crystal structures, side chains <br>
> could be placed with reasonable confidence, particularly smaller ones. <br>
> Should I restrict to poly-ala model or take the advantage of crystal <br>
> structures and include side chains also?<br>
><br>
> 2) When I tried to refine with side chains, longer side chains like K, <br>
> R, Q, E which have weaker density tend to bend towards the backbone <br>
> density. Is there a way to prevent this happening?<br>
><br>
> Suggestions/comments from the community would be highly appreciated.<br>
><br>
> Thanks !<br>
><br>
> Ashu<br>
<br>
</blockquote></div>