Q: How do I choose the correct arguments for the amask argument in refine? I see that it ultimately is used to run the amask2 argument in proc3d, but I'm still a little unsure as do how to choose the parameters. I take it that the 'r' is the approximate radius where the density of the molecule falls off, and thr is the value of ...? And what is N? Thank you.
A: Automasking performs a procedure very much like solvent flattening, and can be critical for obtaining an optimal reconstruction. The only case where it should be avoided is where there are flexible or low density structures sticking out of your map which you want to insure you don't accidentally remove.
Here is the procedure for determining automask parameters. There is also some information in the proc3d manual page under automask2= :
Visualize your map in Chimera or some other isosurface visualization program. Pick an isosurface threshold at which your map 'looks good', then lower the threshold a little below that point. You want to avoid lowering it so much that any blobs of noise outside the map become connected to the bulk of the map itself. However, you also need to insure that the density representing your protein is fully interconnected. Typically 0.1-0.3 below the 'good' threshold works well. This isosurface threshold is the SECOND parameter for amask=.
The first parameter for amask= is a radius in pixels. This should specify the size of a sphere which JUST BARELY comes into contact with the inside edge of your model. This can be done by trial and error. In many cases 2-3 pixels will be fine, but if your map is hollow, you will need a larger value.
The third parameter specifies the width in pixels of a soft mask shell which goes around the outside of your particle. It helps insure that masking doesn't produce any resolution exaggeration. Typically this number is box_size_in_pixels/10, but you can go +-50% from this value.
To test your 3 parameters:
proc3d my_map.mrc test_map.mrc automask2=<r>,<threshold>,<width>
then: v4 my_map.mrc test_map.mrc
this will let you visualize the map and satisfy yourself that you aren't removing anything inappropriate. When you have the parameters you like, in refine, add: amask=<r>,<threshold>,<width>