Hi,
I am working on a ‘small’ (~85kDa) membrane protein with low snr particles (~270k). NU refinement doesn’t work well (~6-7A), however using local refinement excluding a flexible domain (~20kDa) appears to ‘work’ (excluded by mask, no particle subtraction). My issue is what I believe to be overfitting - while I see density improvements in the tm regions down to ~3.5A, the loop regions gets overfitted to a point when maps are not interpretable (refinement keeps goooooing until tm regions break down too, <3A).
I’ve varied mask dilation/padding (0-5 & 10-30), turned on gaussian priors (3/2). When I ‘restrict’ refinement sufficiently with these parameters (i.e. no overfitting) the refinement stops @ ~4-4.5A. My issue is that there is clearly high res information beyond this resolution in the tm regions…
Anyone had a similar issues before? Thoughts? Suggestions?
Cheers,
Mika