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To be honest, I'm a little surprised by this discussion, as it's part of most basic CryoEM intro courses. While Ed is correct for helices, of course, and while having all possible side views does, indeed, yield a complete data set, there is a problem in the
case of single particle analysis. In helical reconstruction, once the symmetry is known, there is a relationship between linear position along the length of the helix and orientation about the helical axis.
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<div class="">In the case of single particle analysis, with only pure 'side views' (perpendicular to some axis), there is no information available to accurately determine the angle about the symmetry axis, since the only common-line the projections share lies
on the helical axis. While there are approaches to come up with reasonable results (for example the 'sidewinder' program developed by Penczek). To achieve good particle orientations requires at least having reasonably good tilted views to anchor the orientations.
"top" views are not required, of course, but if _only_ pure side views are available you will definitely run into problems. You might get by with 10-20 degree tilted views, but it's best if you have a population that goes up to at least ~45 degrees.
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Steven Ludtke, Ph.D. <<a href="mailto:sludtke@bcm.edu" class="">sludtke@bcm.edu</a>> Baylor College of Medicine <br class="">
Charles C. Bell Jr., Professor of Structural Biology<br class="">
Dept. of Biochemistry and Molecular Biology (<a href="http://www.bcm.edu/biochem" class="">www.bcm.edu/biochem</a>)<br class="">
Academic Director, CryoEM Core (<a href="http://cryoem.bcm.edu" class="">cryoem.bcm.edu</a>)<br class="">
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<div class="">On Mar 25, 2020, at 12:57 PM, Egelman, Edward H (ehe2n) <<a href="mailto:egelman@VIRGINIA.EDU" class="">egelman@VIRGINIA.EDU</a>> wrote:</div>
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<span style="font-size: 12pt;" class="">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 class=""></o:p></span></div>
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<span style="font-size: 12pt;" class="">Regards,<o:p class=""></o:p></span></div>
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<span style="font-size: 12pt;" class="">Ed<o:p class=""></o:p></span></div>
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<span style="font-size: 12pt;" class=""><o:p class=""> </o:p></span></div>
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<span style="font-size: 10.5pt;" class="">Edward Egelman<o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class="">Harrison Distinguished Professor<o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class="">Department of Biochemistry and Molecular Genetics<o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class="">University of Virginia<o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class="">phone: 434-924-8210<o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class=""><a href="mailto:egelman@virginia.edu" style="color: blue; text-decoration: underline;" class="">egelman@virginia.edu</a><o:p class=""></o:p></span></div>
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<span style="font-size: 10.5pt;" class=""><a href="https://urldefense.proofpoint.com/v2/url?u=http-3A__www.people.virginia.edu_-7Eehe2n&d=DwMGaQ&c=ZQs-KZ8oxEw0p81sqgiaRA&r=GWA2IF6nkq8sZMXHpp1Xpg&m=bz-LM9mat-VnrSfeo_Oir-L1-yt-iATfenf6mPMKeW8&s=HPp7p-R9ytPZgJZl8ijWxuo4wou_TpDhggKfNqU65R8&e=" style="color: blue; text-decoration: underline;" class="">www.people.virginia.edu/~ehe2n</a></span><span style="font-size: 12pt;" class=""><o:p class=""></o:p></span></div>
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<span style="font-size: 12pt;" class=""><o:p class=""> </o:p></span></div>
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<b class=""><span style="font-size: 12pt;" class="">From:<span class="Apple-converted-space"> </span></span></b><span style="font-size: 12pt;" class="">3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" style="color: blue; text-decoration: underline;" class="">3dem-bounces@ncmir.ucsd.edu</a>>
on behalf of Tom Calcraft <<a href="mailto:tom.calcraft@crick.ac.uk" style="color: blue; text-decoration: underline;" class="">tom.calcraft@crick.ac.uk</a>><br class="">
<b class="">Date:<span class="Apple-converted-space"> </span></b>Wednesday, March 25, 2020 at 1:38 PM<br class="">
<b class="">To:<span class="Apple-converted-space"> </span></b>Basil Greber <<a href="mailto:basilgreber@gmx.net" style="color: blue; text-decoration: underline;" class="">basilgreber@gmx.net</a>><br class="">
<b class="">Cc:<span class="Apple-converted-space"> </span></b>3dem <<a href="mailto:3dem@ncmir.ucsd.edu" style="color: blue; text-decoration: underline;" class="">3dem@ncmir.ucsd.edu</a>><br class="">
<b class="">Subject:<span class="Apple-converted-space"> </span></b>Re: [3dem] Membrane protein structure: orientation issue<o:p class=""></o:p></span></div>
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Hi,<o:p class=""></o:p></div>
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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 class="">
Alsahafi et al (2019) An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity<o:p class=""></o:p></div>
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<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__www.sciencedirect.com_science_article_pii_S1931312819301143-3Fvia-3Dihub-23bib6&d=DwMGaQ&c=ZQs-KZ8oxEw0p81sqgiaRA&r=GWA2IF6nkq8sZMXHpp1Xpg&m=bz-LM9mat-VnrSfeo_Oir-L1-yt-iATfenf6mPMKeW8&s=E9PI4B_BI0Q5ONQn2-O9IPczL6KfAnhOuno6GUJcQXA&e=" style="color: blue; text-decoration: underline;" class="">https://www.sciencedirect.com/science/article/pii/S1931312819301143?via%3Dihub#bib6</a><o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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The orientation distribution in the paper supplement shows that it is almost completely composed of side views.<o:p class=""></o:p></div>
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<o:p class=""> </o:p></div>
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All the best <o:p class=""></o:p></div>
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Tom<o:p class=""></o:p></div>
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On 25 Mar 2020, at 16:23, Basil Greber <<a href="mailto:basilgreber@gmx.net" style="color: blue; text-decoration: underline;" class="">basilgreber@gmx.net</a>> wrote:<o:p class=""></o:p></div>
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<span style="font-size: 9pt; font-family: Helvetica;" class="">I am also confused by this. Shouldn’t a tomographic series (180 degrees worth of side views) do it for a C1 particle?<br class="">
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Basil<br class="">
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<span style="font-size: 9pt; font-family: Helvetica;" class="">Am 25.03.2020 um 00:06 schrieb Philip Köck <<a href="mailto:koeck@kth.se" style="color: blue; text-decoration: underline;" class="">koeck@kth.se</a>>:<br class="">
<br class="">
Why do you say that the symmetry has to be at least C3?<br class="">
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All the best,<br class="">
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Philip<br class="">
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Från: 3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu" style="color: blue; text-decoration: underline;" class="">3dem-bounces@ncmir.ucsd.edu</a>> för Dmitry Lyumkis <<a href="mailto:dlyumkis@salk.edu" style="color: blue; text-decoration: underline;" class="">dlyumkis@salk.edu</a>><br class="">
Skickat: den 25 mars 2020 07:07<br class="">
Till: Jacopo Marino<br class="">
Kopia:<span class="Apple-converted-space"> </span><a href="mailto:3dem@ncmir.ucsd.edu" style="color: blue; text-decoration: underline;" class="">3dem@ncmir.ucsd.edu</a><br class="">
Ämne: Re: [3dem] Membrane protein structure: orientation issue<br class="">
<br class="">
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 class="">
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Dmitry Lyumkis<br class="">
Assistant Professor<br class="">
The Salk Institute for Biological Studies<br class="">
10010 North Torrey Pines Road, La Jolla, CA 92037<br class="">
T: 858-453-4100 x1155<br class="">
E:<span class="Apple-converted-space"> </span><a href="mailto:dlyumkis@salk.edu" style="color: blue; text-decoration: underline;" class="">dlyumkis@salk.edu</a><br class="">
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On Mar 24, 2020, at 10:53 PM, Jacopo Marino <<a href="mailto:jacopo.marino@psi.ch" style="color: blue; text-decoration: underline;" class="">jacopo.marino@psi.ch</a>> wrote:<br class="">
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How much do you really need top and bottom views for 3D reconstruction ? Have you tried ?<br class="">
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On 25.03.2020 06:48, Saumya Verma Bajaj wrote:<br class="">
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Dear all,<span class="apple-converted-space"> </span><br class="">
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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 class="">
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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 class="">
<br class="">
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 class="">
<br class="">
Any suggestions to salvage this set of data will be very helpful.<span class="apple-converted-space"> </span><br class="">
<br class="">
Thank you.<br class="">
Best regards,<br class="">
Saumya<span class="apple-converted-space"> </span><br class="">
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