[3dem] Membrane protein structure: orientation issue
Sjors Scheres
scheres at mrc-lmb.cam.ac.uk
Thu Mar 26 06:15:49 PDT 2020
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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