[3dem] Membrane protein structure: orientation issue

Wed Mar 25 12:31:14 PDT 2020

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.

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. <sludtke at bcm.edu<mailto:sludtke at bcm.edu>>                      Baylor College of Medicine
Charles C. Bell Jr., Professor of Structural Biology
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On Mar 25, 2020, at 12:57 PM, Egelman, Edward H (ehe2n) <egelman at VIRGINIA.EDU<mailto:egelman at VIRGINIA.EDU>> wrote:

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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.
Regards,
Ed

Edward Egelman
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From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Tom Calcraft <tom.calcraft at crick.ac.uk<mailto:tom.calcraft at crick.ac.uk>>
Date: Wednesday, March 25, 2020 at 1:38 PM
To: Basil Greber <basilgreber at gmx.net<mailto:basilgreber at gmx.net>>
Cc: 3dem <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] Membrane protein structure: orientation issue

Hi,

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:
Alsahafi et al (2019) An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity
https://www.sciencedirect.com/science/article/pii/S1931312819301143?via%3Dihub#bib6<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=>

The orientation distribution in the paper supplement shows that it is almost completely composed of side views.

All the best
Tom

On 25 Mar 2020, at 16:23, Basil Greber <basilgreber at gmx.net<mailto:basilgreber at gmx.net>> wrote:

I am also confused by this. Shouldn’t a tomographic series (180 degrees worth of side views) do it for a C1 particle?

Basil

Am 25.03.2020 um 00:06 schrieb Philip Köck <koeck at kth.se<mailto:koeck at kth.se>>:

Why do you say that the symmetry has to be at least C3?

All the best,

Philip

Från: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> för Dmitry Lyumkis <dlyumkis at salk.edu<mailto:dlyumkis at salk.edu>>
Skickat: den 25 mars 2020 07:07
Till: Jacopo Marino
Kopia: 3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
Ämne: Re: [3dem] Membrane protein structure: orientation issue

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.

Dmitry Lyumkis
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On Mar 24, 2020, at 10:53 PM, Jacopo Marino <jacopo.marino at psi.ch<mailto:jacopo.marino at psi.ch>> wrote:

How much do you really need top and bottom views for 3D reconstruction ? Have you tried ?

On 25.03.2020 06:48, Saumya Verma Bajaj wrote:

Dear all,

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.

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.

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.

Any suggestions to salvage this set of data will be very helpful.

Thank you.
Best regards,
Saumya
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