[3dem] Membrane protein structure: orientation issue

[Sjors Scheres]

Hi Saumya,

As others have remarked too: it actually doesn't look so bad and you 
don't really need the top views. I would try and combine particles from 
basically all the 2D classes that you show (possibly excluding some from 
the bottom 2 rows, but that will not matter much) for a 3D initial model 
generation (if you don't have a model yet) and then refine or 
3d-classify with the expected symmetry (C4?). You might be surprised.

HTH,

Sjors


On 25/03/2020 05:48, 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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