[3dem] Membrane protein structure: orientation issue

Wed Mar 25 10:57:56 PDT 2020

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

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From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Tom Calcraft <tom.calcraft at crick.ac.uk>
Date: Wednesday, March 25, 2020 at 1:38 PM
To: Basil Greber <basilgreber at gmx.net>
Cc: 3dem <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://www.sciencedirect.com/science/article/pii/S1931312819301143?via=ihub#bib6>

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
Assistant Professor
The Salk Institute for Biological Studies
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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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