I have successfully ran local refinements without particle subtraction on my map (subunit A and B) and used ‘combine focused map’ in PHENIX to obtain a composite map, which looks very nice. Now I want to post-process/sharpen the composite map. What is the proper way to go about it? Should I sharpen the composite map or on each individual subunit before combining them?
Since you are already using PHENIX, I recommend using PHENIX autosharpen or ResolveEM (density modification) on your composite maps. You can also try making a composite of the sharpened maps from the refinement, which use the traditional Guinier analysis to determine a single b-factor. Then use whichever of these 3 approaches gives you the most interpretable map.
I also recommend you try loading the maps in Chimera(X) and combining them with
vop max ... as an alternative compositing approach. Again, use whichever method is more interpretable.
Thank you so much for your suggestion! I will definitely try your suggestions on sharpening in PHENIX and compositing maps in ChimeraX.
I have a quick, naive question regarding post-processing/sharpening a composite map. ResolveEM in PHENIX seems to require two half maps. Do you know a way around it to use ResolveEM (or similar tools that require two half maps) on a composite map? Or when two half maps are required, would sharpening local-refined maps before compositing them the only option?
I forget if you can make ResolveEM run without half-maps. I know there’s an option to also provide the full map. Anyway, you may also want to estimate local resolution in your composite map in the future, and this problem has the same solution: simply make composite half-maps. That is, composite the two A half-maps in each local refinement, and the two B half-maps, and then use these as composite half-maps for any processing that requires two half-maps.
As long as your local refinements have the same half subsets (which is the default behavior), this is fine to do.
I didn’t think of making composite half maps, which is a great idea. I will also estimate local resolution by using composite half maps. Thank you very much for the help!
No problem. Would you mind posting a picture of the FSC curve you get from the composite half-maps? I just want to see if it looks completely as expected or not.
I did what you suggested, and it looks bit weird.
In Chimera X, I loaded original half A (J1321) and two local-refined half A’s (J1360 and J1363) and composited them using vop maximum #1 #2 #3. I repeated the procedure to make a composite B map. I imported two composite half maps and estimated the local resolution (default parameters at 0.5 FSC threshold) using composite half A and B as well as mask refine from J1321. I referenced Local res. estimate with imported map error. I have attached the FSC curves of all relevant refinement jobs.
J1321 NU-refinement of original reconstruction
J1360 Local Refinement (NEW!) of subunit A (without particle subtraction)
J1363 Local Refinement (NEW!) of subunit B (without particle subtraction)
J1388 Local Resolution Estimation
I have performed the identical procedure on a different map and also obtained very high resolution at 0.5 FSC threshold.
NU-refinement: 2.91 Å
Local Refinement 1: 2.74 Å
Local Refinement 2: 2.76 Å
Local Resolution Estimation: 2.14 Å
Is this the expected behavior?
Hmm, well for the
vop maximum method, you should use only the local refined half-maps, not the original ones. So just
vop max #2 #3 for example. If that doesn’t fix it, could you try without a mask and post the FSC from that?
I re-made the composite maps using only local-refined half-maps (half A’s from J1360 and J1363; half B’s from J1360 and J1363). The resulting FSC from local resolution estimation is as below:
I have done it with and without mask, giving 2.35 Å and 2.37 Å, respectively.
Out of curiosity, does performing local resolution estimation on composite map act as validation method (as in if nothing went wrong)? For publication purposes, it it okay to put FSC curves of each local-refined maps, instead of the composite map?