Not in CCP4, no. And, technically, not in Phenix either. The real-space
refinement in Phenix simply picks peaks in the density and then pulls
nearby atoms toward them. Like a black hole gobbling up nearby planets.
It took me a while to realize that! If you manage to turn off geometry
restraints (as I eventually did) all the atoms end up on top of each
other. Might seem like a horrible idea, but for poor resolution data and
reasonably good geometry restraints it has a high radius of convergence
and is incredibly fast when compared to "real real-space refinement".
Refining against map voxels directly is a very very slow process.
But, if you real really want to do real real-space, then I suppose coot
is doing that? I'm actually not sure.
The isolde suggestion already made is an excellent one. The hardest
part of that is getting the right version of chimeraX working. But,
once you've done that its pretty straightforward.
One program that has not been mentioned, but does "real real" space
refinement is: "rsref"
https://chapman.missouri.edu/wp-content/uploads/sites/2/software/rsref/html/rsref_doc.html
It is not too hard to install and use. I can't say I've gotten results
appreciably different from reciprocal-space refinement, and that led me
to ask myself why exactly I thought it would be different. The Fourier
transform is symmetric after all. But I do expect that if you have
unmodeled regions, such as big, spiky metals, or large tracts of
disordered, ropy stuff, then localizing the refinement could be beneficial.
Now, of course, you can also do localized refinement in reciprocal space
by just smoothing out parts of the map that are "uninteresting". The
vast area of noise around the protein in a cryoEM map, for example, is
perhaps a candidate for noise suppression. The only trick is how to
suppress noise without creating systematic error. For example, if your
model does not have "bulk solvent" then this area will be modeled as
vacuum, but if you simply set the map voxel values to 0.00, you will
have effectively created more bulk solvent, not eliminated it. This is
because 0.00 is usually the average voxel value, not the "vacuum level".
Then there is the "edge" between the modified and unmodified areas.
Unless you smooth it in some way this edge will be very sharp and
therefore have significant Fourier coefficients at a wide range of
resolutions. So, if you are not careful, your "noise suppression" can
create a lot more error than it eliminates.
As for what to do? The scale factor given to the "bulk solvent" model
is perhaps the best value to use to replace the "bulk" solvent region.
The bulk solvent mask itself, ranging from 0 to 1, might also be a
reasonable weighting function for combining your original map with a
single-valued map. That is, don't change the protein, but flatten the
solvent. You can get this map out of refmac using the MSKOUT feature.
You then smooth it in reciprocal space by applying refmac's best-fit
solvent B factor using sfall and fft, then finally scale it with
mapmask. I should admit, however, that I have not tried this in a
while. Let me know if it works!
HTH
-James Holton
MAD Scientist
On 7/29/2020 8:20 AM, Schreuder, Herman /DE wrote:
Dear BB,
I would like to do a real real-space-refinement of a protein against a
cryo-EM map; not the mtz-based Refmac approach. A quick internet
search produced a lot of Phenix hits, but little ccp4 hits. Does
somebody know how to do this using ccp4 programs, or has someone a
Coot script to do this?
Thank you for your help!
Herman
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