[3dem] Membrane protein structure: orientation issue

[Bruno KLAHOLZ]

Dear Saumya,

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).

Best,

Bruno



From: 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] On Behalf Of Jacopo Marino
Sent: 25 March 2020 06:53
To: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Membrane protein structure: orientation issue


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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Dr. Jacopo Marino

Laboratory of Biomolecular Research

Paul Scherrer Institute

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Bruno P. Klaholz
Centre for Integrative Biology
Department of Integrated Structural Biology
Institute of Genetics and of Molecular and Cellular Biology
IGBMC - UMR 7104 - U 1258
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BP 10142
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websites:
http://igbmc.fr/Klaholz
http://www.igbmc.fr/grandesstructures/cbi<http://www.igbmc.fr/grandesstructures/cbi/>
http://frisbi.eu<http://frisbi.eu/>
http://instruct-eric.eu


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