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<p class="MsoNormal"><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif;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-fareast-language:EN-US">Dear Saumya,<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-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-fareast-language:EN-US">the first impression from the 2D class averages is that they are not centered, some even go close to the mask on the
edge (true both for the protein core and the transmembrane region). It would be worth checking the particle selection step and make sure particles are reasonably well centered. This will help getting better views for the more rare orientations. Once this is
done, try removing some of the predominant views to reduce preferential view effects (but in fact the angular distribution looks still pretty good as there are also intermediate views).<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-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-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-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-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-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-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-fareast-language:EN-US"><o:p> </o:p></span></font></p>
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<p class="MsoNormal"><b><font size="2" face="Calibri"><span lang="EN-US" style="font-size:11.0pt;font-family:"Calibri",sans-serif;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">
3dem [mailto:3dem-bounces@ncmir.ucsd.edu] <b><span style="font-weight:bold">On Behalf Of
</span></b>Jacopo Marino<br>
<b><span style="font-weight:bold">Sent:</span></b> 25 March 2020 06:53<br>
<b><span style="font-weight:bold">To:</span></b> 3dem@ncmir.ucsd.edu<br>
<b><span style="font-weight:bold">Subject:</span></b> Re: [3dem] Membrane protein structure: orientation issue<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>
<p><tt><font size="2" face="Courier New"><span style="font-size:10.0pt">How much do you really need top and bottom views for 3D reconstruction ? Have you tried ?</span></font></tt><o:p></o:p></p>
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<p class="MsoNormal"><font size="3" face="Times New Roman"><span style="font-size:12.0pt">On 25.03.2020 06:48, Saumya Verma Bajaj wrote:<o:p></o:p></span></font></p>
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<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Dear all,</span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif"> </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">I am trying to solve the structure of a tetrameric membrane protein complex, with the protein embedded in detergent micelle
(0.05% GDN) and a soluble accessory protein attached to it. </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif"> </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Following 2D classification, while the side views and oblique views are easily visible, and the top/bottom are very few
(~1-2%) (please see attached image) and get further diminished in subsequent rounds of 2D classification. </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif"> </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">While we are trying different grid types to overcome the orientation problem at the sample level, I was wondering if there
are certain tweaks we can make to the analysis parameters (particle picking, box size, mask, ctf etc.) to enhance the signal of protein embedded within a micelle in the current data set. We are using Relion3.1 for SPA.</span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif"> </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Any suggestions to salvage this set of data will be very helpful.</span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif"> </span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Thank you.</span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Best regards,</span></font><o:p></o:p></p>
<p class="MsoNormal" style="text-align:justify"><font size="3" face="Arial"><span style="font-size:12.0pt;font-family:"Arial",sans-serif">Saumya</span></font><o:p></o:p></p>
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<p class="MsoNormal"><font size="3" face="Times New Roman"><span style="font-size:12.0pt"><br>
<br>
<o:p></o:p></span></font></p>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">_______________________________________________<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">3dem mailing list<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt"><a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt"><a href="https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem">https://mail.ncmir.ucsd.edu/mailman/listinfo/3dem</a><o:p></o:p></span></font></pre>
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<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">-- <o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">Dr. Jacopo Marino<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">Laboratory of Biomolecular Research<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">Paul Scherrer Institute<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">OFLB/005<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">5232 Villigen PSI, Switzerland<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">tel: +41 56 310 5484 (or +41 56 310 5777)<o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt">e-mail: <a href="mailto:jacopo.marino@psi.ch">jacopo.marino@psi.ch</a><o:p></o:p></span></font></pre>
<pre><font size="2" face="Courier New"><span style="font-size:10.0pt"><o:p> </o:p></span></font></pre>
<p class="MsoNormal"><font size="3" face="Times New Roman"><span style="font-size:12.0pt"><o:p> </o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">###########################################################################<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">Bruno P. Klaholz<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">Centre for Integrative Biology<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">Department of Integrated Structural Biology<br>
Institute of Genetics and of Molecular and Cellular Biology<br>
IGBMC - UMR 7104 - U 1258<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">1, rue Laurent Fries<br>
BP 10142 <br>
67404 ILLKIRCH CEDEX<br>
FRANCE<o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt">websites:<br>
<a href="http://igbmc.fr/Klaholz" target="_blank"><font color="#3a2eb5"><span style="color:#3A2EB5">http://igbmc.fr/Klaholz</span></font></a><o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt"><a href="http://www.igbmc.fr/grandesstructures/cbi/" target="_blank">http://www.igbmc.fr/grandesstructures/cbi</a><o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt"><a href="http://frisbi.eu/" target="_blank">http://frisbi.eu</a><o:p></o:p></span></font></p>
<p class="MsoNormal"><font size="2" face="Times New Roman"><span style="font-size:11.0pt"><a href="http://instruct-eric.eu">http://instruct-eric.eu</a><o:p></o:p></span></font></p>
<pre><font size="2" face="Calibri"><span style="font-size:11.0pt;font-family:"Calibri",sans-serif"><o:p> </o:p></span></font></pre>
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