Hey @LTP,
Both subtraction and local refinement should work as usual with downsampled data. If you’re seeing all of the FSC curves (even the unmasked FSC) quite high all the way out to the max resolution, it could likely be a genuine resolution increase, in which case yes, moving to the unbinned data could bump up the resolution more. You would have to re-do signal subtraction though – but you can use the same alignments from the unbinned data by overriding the “blob” input using the job builder’s low-level results feature.
The other main causes of unexpectedly high FSC curves could be:
- Duplicated particles crossing the GS split, which you do have to be careful of when working with symmetry expanded particles (from above).
- Using binary / not-soft masks for any refinements – this often makes the loose and tight masked FSC curves wacky (see Oli’s post where we figured out that the cause of his strange FSCs here was the binary mask)
Best,
Michael