[3dem] Masked 3D classification job without alignment to classify promoters of a trimeric protein in different conformations

[Basil Greber]

I am not sure your symmetry expansion will do what you want when starting from a C1 refinement (unless you made sure your reference map is perfectly aligned with the symmetry axis). If you want to do symmetry expansion, you may need a C3 refinement.

That said, if your C1 refinement can distinguish the two conformations, you probably don’t need symmetry expansion in the first place. Just run 3 classifications with the mask in each protomer position to classify each position. Given your description, this is probably the better approach.

Good luck,

Basil


> Am 05.01.2023 um 12:56 schrieb Shashank Khare <shashank.khare at u-bordeaux.fr>:
> 
> Hi,
>  
> I am working on a membrane protein which forms a trimer. Interestingly, the 2 protomers are in outward conformation while the 3rd protomer is in inward conformation. I am trying to classify the individual protomers using masked 3D classification job without alignment in relion 4 beta. I am using refined particles (C1) as an input for this job.
>  
> There are following pipelines I have tried so far,
>  
> 1.  3D Auto-Refine (C1)  Symmetry expansion (C3)  Signal subtraction (masking the 3rd protomer in inward conformation  3D Classification (mask of 3rd Protomer).
> 2.  3D Auto-Refine(C1)  Symmetry expansion (C3)  Signal subtraction (invert map created using the 3rd protomer in inward conformation  3D Classification (mask of 3rd Protomer).
> 3.  3D Auto-Refine (C1)  Symmetry expansion (C3)   3D Classification (mask of 3rd Protomer).
>  
> I have also explored different T values from 10-40 and k values from 4-8. But the output of all of these jobs is only in inward conformation which corresponds to 3rd protomer.
>  
> It would be really helpful if someone can guide me with the pipeline here.
>  
> Regards,
> Shashank. 
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