I have some questions about the 3D variability analysis parameters and interpretation of the results.
I got impression that the ‘filter resolution’ should be lower than the consensus resolution and on the range of the extent of the movement you would expect to see. My impression is to set larger filter resolution numbers for larger movement? It is not stated in the turorial webpage and I can’t find where I got the impression. Please correct me if I am wroing.
I have Cryo-EM structures of the enzyme alone (3.61A from non-uniform refinement, 82K particles, 110kD) and in complex with the substrate (3.96A from non-uniform refinement, 60K particles, 110+25=135kD). I didn’t find any subsets with different conformation from either datasets by rounds of ab-initio and heterogeneous until two identical volumes were re-constructed.
I would like to see if the enzyme is more stable when binding to substrate.
I tried to set up ‘filter resolution’ to 4,5,7,10A for the enzyme alone structure; and saw some interesting movement (simple mode in 3D display; a 37kD domain tilting against a 73kD domain) when ‘filter resolution’ was set to 10A. Other settings of 4,5,7 did not generate obvious movements. Does this mean the movement is too big or too weak to be identified when the filter resolution is set too small? We have a crystal structure of the enzyme which shows a large domain tilting when aligned with the consensus EM structure model. Therefore, we would expect EM structures would capture some flexibility f
of the enzyme.
I tried to set up ‘filter resolution’ to 4,5,10A for the enzyme+substrate structure, and it generated obvious and similar (62kD domain tilting against 73 domain) movement when it was set to 5A and 10A.
It is difficult to tell which movement is more significant by eyes. Is there a quantitative way to show the enzyme in which state has higher flexibility since we see flexibility in both structures? Dose the different effective settings of ‘filter resolution’ implicate anything? Are the volumes for all the ‘frames’ saved somewhere so I can fit in model and align models?
Thanks very much for reading my post and any comments would be appreciated.