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

[Shashank Khare]

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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