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<div class="WordSection1">
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">Hello,<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">preferential views can give quite significant distorsions in cryo-EM maps.<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">What is your current Euler angle distribution plot?<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">If preferential views are significant and/or if you see fine stripes in the
sections of your 3D reconstruction you can try to remove some of the most prominent preferential views (i.e. remove those particles), but be careful to keep the more rare views (these tend to get removed because the quality of re-projections is not as good
/ is more noisy, which may disturb structure (cryo-EM map) refinement).<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">Best,<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US">Bruno<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-bidi-font-family:"Times New Roman";mso-fareast-language:EN-US"><o:p> </o:p></span></font></p>
<div>
<div style="border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0cm 0cm 0cm">
<p class="MsoNormal"><b><font size="2" face="Calibri"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US;font-weight:bold">From:</span></font></b><font size="2" face="Calibri"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US">
phenixbb-bounces@phenix-online.org [mailto:phenixbb-bounces@phenix-online.org] <b>
<span style="font-weight:bold">On Behalf Of </span></b>Carlos HENRIQUE FERNANDES<br>
<b><span style="font-weight:bold">Sent:</span></b> 25 May 2021 18:35<br>
<b><span style="font-weight:bold">To:</span></b> Tom Terwilliger <tterwilliger@newmexicoconsortium.org><br>
<b><span style="font-weight:bold">Cc:</span></b> phenixbb <phenixbb@phenix-online.org><br>
<b><span style="font-weight:bold">Subject:</span></b> Re: [phenixbb] Problems with sharpening in Autosharpen<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" face="Times New Roman"><span style="font-size:12.0pt"><o:p> </o:p></span></font></p>
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<p class="MsoNormal" style="margin-bottom:12.0pt"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Hi Tom,
<br>
Thanks for the message.<br>
I did the tests that you suggested to me.<br>
<br>
I runned the autosharpen tutorial data supplied with Phenix and it worked normally. I blurred that map to 300 A**2 and the resulting map was indeed similar to my map. Finally, I applied the Autosharpen to the blurred map and it was applied a B-sharpen of 222.44
A**2. So, everything worked normally.<br>
<br>
I was thinking that may be disturbing the sharpening is that I have a clearly preferred protein orientation in single-particle analysis. In my data set, the side views are more abundant than the rest of views in my 2D classes (and the number of particles in
side view 2D classes are much more larger than in the rest of views 2D classes). What do you think?<br>
<br>
Thanks again for the help.<br>
All the best,<br>
Carlos.<o:p></o:p></span></font></p>
<div class="MsoNormal" align="center" style="text-align:center"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">
<hr size="2" width="100%" align="center" id="zwchr">
</span></font></div>
<div>
<p class="MsoNormal" style="mso-outline-level:1"><b><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black;font-weight:bold">De:
</span></font></b><font color="black" face="Arial"><span style="font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">"Tom Terwilliger" <<a href="mailto:tterwilliger@newmexicoconsortium.org">tterwilliger@newmexicoconsortium.org</a>><br>
<b><span style="font-weight:bold">Para: </span></b>"Carlos HENRIQUE FERNANDES" <<a href="mailto:carlos.henrique_fernandes@sorbonne-universite.fr">carlos.henrique_fernandes@sorbonne-universite.fr</a>><br>
<b><span style="font-weight:bold">Cc: </span></b>"phenixbb" <<a href="mailto:phenixbb@phenix-online.org">phenixbb@phenix-online.org</a>>, "Tom Terwilliger" <<a href="mailto:tterwilliger@newmexicoconsortium.org">tterwilliger@newmexicoconsortium.org</a>><br>
<b><span style="font-weight:bold">Enviadas: </span></b>Sexta-feira, 21 de maio de 2021 22:16:57<br>
<b><span style="font-weight:bold">Assunto: </span></b>Re: [phenixbb] Problems with sharpening in Autosharpen<o:p></o:p></span></font></p>
</div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black"><o:p> </o:p></span></font></p>
<div>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Hi Carlos,<o:p></o:p></span></font></p>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Normally if you apply a B-sharpen of 220 to a 3 Å map, the new map should look
really different from the original map (no matter whether you do it yourself or autosharpen does it). So best guess is that something is not happening the way you would like it to be.<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black"><o:p> </o:p></span></font></p>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">A suggestion: run the quick autosharpen tutorial data supplied with Phenix.
Presumably it will work normally. Then tell autosharpen to blur that map to 300 A**2. This map should look something like your map (does it?). Now apply autosharpen to the blurred map. It should apply a B-sharpen of about 250 or so (does it?). And the
new blurred - then - sharpened map should look good.<o:p></o:p></span></font></p>
</div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black"><o:p> </o:p></span></font></p>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Let me know if that doesn't work!<o:p></o:p></span></font></p>
</div>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">All the best,<o:p></o:p></span></font></p>
</div>
<div>
<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Tom T<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black"><o:p> </o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">On Fri, May 21, 2021 at 8:50 AM Carlos HENRIQUE FERNANDES <<a href="mailto:carlos.henrique_fernandes@sorbonne-universite.fr" target="_blank">carlos.henrique_fernandes@sorbonne-universite.fr</a>>
wrote:<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="2" color="black" face="Arial"><span style="font-size:11.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Dear all,
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<br>
I am facing some problems with sharpening in Autosharpen. <br>
My current map is about 3.2 Å resolution, which I obtained after CTF-Refinement and Polishing in Relion.<br>
When I use Autosharpen in default parameters, it tells me that are applied an overall Bsharp applied of 220.34 and a final Biso of 87.42; but very little sharpening is done; I was unable to observe a typical helices sharpening at this value of resolution.
<br>
I did some tests setting progressive Bsharp values (240; 260; 280; 300), but I am still unable to visualize a typical helices sharpening.<br>
Has anyone had problems like that? Any tips? <br>
Thanks in advance,<br>
Carlos.</span></font><font color="black" face="Arial"><span style="font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black"><o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">_______________________________________________<br>
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-- <o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Thomas C Terwilliger<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Laboratory Fellow, Los Alamos National Laboratory<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Senior Scientist, New Mexico Consortium<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">100 Entrada Dr, Los Alamos, NM 87544<o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Email:
<a href="mailto:tterwilliger@newmexicoconsortium.org" target="_blank">tterwilliger@newmexicoconsortium.org</a><o:p></o:p></span></font></p>
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<p class="MsoNormal"><font size="3" color="black" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";color:black">Tel: 505-431-0010<o:p></o:p></span></font></p>
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