Hi Daniel,
I would suggest that the principle to follow is to deposit the map that you
used to interpret the structure, along with the raw data used to get that
map.
The rationale for this is that a reader of your paper would like to be able
to compare map and model and see if they agree with your conclusions, hence
the map you used for interpretation. The reader might also want to check
on the map itself, hence the raw data.
In many cases, there may be more than one map used in interpretation. If
possible, they should probably all be deposited so that others can verify
your analyses.
In your case, I think that would mean that you would want to deposit your
density-modified map as the primary map and the two half-maps produced by
your data processing program and the standard sharpened map as additional
maps. Then if you can, the images (in EMPIAR) as well.
All the best,
Tom T
On Fri, Dec 2, 2022 at 6:23 AM Daniel Larsson
Hi Phenix community,
I’ve been using density modification with some success for my data and am considering the most appropriate way to do model building/refinement and then deposition of my results. Here are some questions/observations:
1) Which map should I refine against (most importantly, do the final minimization using phenix.real_space_refine)? The DM map is the most clear one, so I’m favoring to do it against this one (as long as the geometry/validation is also best for this one).
2) Which map should I deposit as my main map? Is it the autosharpened one or the DM one? Since the DM map looks the best, I want the user who only downloads one map to see this one, so again I favoring this map.
3) Which nominal resolution should I quote for my reconstruction? Since the procedure seems to introduce correlation between the two maps (see point 4 below), it does not seem to be possible to obtain a gold-standard FSC value between two truly independent half-maps. As the DM procedure supposedly actually improves the map (by leveraging knowledge about what solvent and macromolecular maps usually looks like and thereby improves the Fourier coefficients), it feels like it would be misrepresenting the data by quoting the FSC_0.143 from the original reconstruction. Should I use the FSC_0.5 estimated value that phenix.resolve_cryo_em outputs?
4) Which FSC curve should I deposit? When I calculate the FSC between the two density modified half-maps, I get a funny (or rather not so funny) looking curve with a bump at around 3Å and it does not go to zero at Nyquist (which does not agree with the map quality). This must again be due to the induced correlation between the maps, presumably during the masking of solvent and macromolecule density. Phenix.resolve_cryo_em calculates a FSC curve to calculate the FSC_0.5 value, which according to the manual page corrects for the correlation induced between the two maps:
"Density modification introduces some correlations between half-maps due to solvent flattening. This can have a small effect on the resolution estimates obtained with half-map FSC. The resolution estimates provided by the program are corrected for this effect."
But is this curve good enough? I think OneDeposit wants to have the gold-standard FSC curve, right?
5) Which (nowadays mandatory) half-maps should one deposit? Here I would say that the original half-maps before DM makes more sense, since these are less processed. But if one specify these as the half-maps, there will be inconsistencies during validation, since the DM map will look quite different from the average between the two original half-maps. Also the automatic FSC curve will be very different from the deposited one.
So in conclusion, despite what I say above, the most conservative thing would be to deposit the conventionally sharpened map as my main map and use the original half-maps for calculating the FSC-curve and use the 0.143 threshold from this curve to estimate the resolution. The DM map can be used during rebuilding/refinement, but I think final minimization should be against the main map during deposition (with restraints if the geometry gets worse). The DM map can be deposited as an auxiliary map.
Do the community and the developers agree?
Regards, Daniel
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