Hi all,
I am currently working on a small transporter reconstituted in MSP1D1/POPC-POPG nanodiscs, with a nanobody bound to the C-terminus to facilitate particle alignment.
The 2D classifications look promising. The α-helices are clear and visible in the ab initio reconstruction, however they disappear after hetero-refinement and/or NU-refinement. It seems that the MSP proteins dominate the alignment. I am wondering whether the nanodiscs could make the transporter more flexible, resulting in poor alignment. Would it make sense to mask out the MSP proteins? Cause I have been trying to do so, but it is difficult to obtain a map with continuous α-helical density at the moment.
I have attached the results from 2D classification, ab-initio reconstruction and hetero-refinement. Have I made any mistakes during processing, or would it be better to use detergents for this protein instead? I would appreciate any suggestions.
Many thanks,
C
Parameters for Ab-Initio:
Initial resolution (Angstroms): 20→10→8
Maximum resolution (Angstroms): 8→6→4.5
Number of initial iterations: 300
Number of final iterations: 500
Initial minibatch size: 1000
Final minibatch size: 3000 or 5000
Class similarity: 0.1→0.02
Parameters for Heterogeneous refinement:
Spherical mask diameter (A): 160
Max alignment resolution (A): 15
