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<p class="MsoNormal"><span style="font-size:12.0pt">But this is done all the time in helical reconstruction, to resolutions better than 3.0 Å. Having all projections of side-views is a single-axis tilt series which yields all information needed in the absence
of any symmetry.<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt">Regards,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:12.0pt">Ed<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:10.5pt;color:black">University of Virginia<o:p></o:p></span></p>
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<p class="MsoNormal"><b><span style="font-size:12.0pt;color:black">From: </span></b><span style="font-size:12.0pt;color:black">3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Tom Calcraft <tom.calcraft@crick.ac.uk><br>
<b>Date: </b>Wednesday, March 25, 2020 at 1:38 PM<br>
<b>To: </b>Basil Greber <basilgreber@gmx.net><br>
<b>Cc: </b>3dem <3dem@ncmir.ucsd.edu><br>
<b>Subject: </b>Re: [3dem] Membrane protein structure: orientation issue<o:p></o:p></span></p>
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<p class="MsoNormal">Hi,<o:p></o:p></p>
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<p class="MsoNormal">There has been a paper where they applied this logic to get a ~13Å reconstruction of the HIV Env spikes from intact virus, without imposing symmetry: <br>
Alsahafi et al (2019) An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity<o:p></o:p></p>
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<p class="MsoNormal"><a href="https://www.sciencedirect.com/science/article/pii/S1931312819301143?via=ihub#bib6">https://www.sciencedirect.com/science/article/pii/S1931312819301143?via%3Dihub#bib6</a><o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">The orientation distribution in the paper supplement shows that it is almost completely composed of side views.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal">All the best <o:p></o:p></p>
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<p class="MsoNormal">Tom<o:p></o:p></p>
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<p class="MsoNormal">On 25 Mar 2020, at 16:23, Basil Greber <<a href="mailto:basilgreber@gmx.net">basilgreber@gmx.net</a>> wrote:<o:p></o:p></p>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:Helvetica">I am also confused by this. Shouldn’t a tomographic series (180 degrees worth of side views) do it for a C1 particle?<br>
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Basil<br>
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<p class="MsoNormal"><span style="font-size:9.0pt;font-family:Helvetica">Am 25.03.2020 um 00:06 schrieb Philip Köck <<a href="mailto:koeck@kth.se">koeck@kth.se</a>>:<br>
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Why do you say that the symmetry has to be at least C3?<br>
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All the best,<br>
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Philip<br>
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Från: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>> för Dmitry Lyumkis <<a href="mailto:dlyumkis@salk.edu">dlyumkis@salk.edu</a>><br>
Skickat: den 25 mars 2020 07:07<br>
Till: Jacopo Marino<br>
Kopia: <a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a><br>
Ämne: Re: [3dem] Membrane protein structure: orientation issue<br>
<br>
Assuming this is a symmetric membrane protein with at least 3-fold rotational symmetry, you don’t need the top and bottom views to fully sample Fourier space and arrive at a high-quality reconstruction. Side-views of a rotationally symmetric particle are sufficient,
and the reconstruction will be complete.<span class="apple-converted-space"> </span><br>
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On Mar 24, 2020, at 10:53 PM, Jacopo Marino <<a href="mailto:jacopo.marino@psi.ch">jacopo.marino@psi.ch</a>> wrote:<br>
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How much do you really need top and bottom views for 3D reconstruction ? Have you tried ?<br>
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<br>
On 25.03.2020 06:48, Saumya Verma Bajaj wrote:<br>
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Dear all,<span class="apple-converted-space"> </span><br>
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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. <br>
<br>
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. <br>
<br>
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 class="apple-converted-space"> </span><br>
<br>
Any suggestions to salvage this set of data will be very helpful.<span class="apple-converted-space"> </span><br>
<br>
Thank you.<br>
Best regards,<br>
Saumya<span class="apple-converted-space"> </span><br>
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