Dear Ian,
This has been discussed in a review and related articles by Brian
Matthews and Liljun Liu:
Matthews BW, Liu L. A review about nothing: are apolar cavities in proteins
really empty? Protein Sci. 2009 Mar;18(3):494-502. doi: 10.1002/pro.61. Review.
PubMed PMID: 19241368; PubMed Central PMCID: PMC2760356.
Daniel
Le 16/06/2014 11:32, Ian Tickle a écrit :
Dear James
You seem to be discounting the possibility of a true vacuum inside a
structure, which is obviously not the same thing as 'something' (bulk
solvent or whatever). I accept that this is unlikely in the case of
ligand binding sites exposed to solvent, or indeed any site on the
outer surface of the molecule, since any vacuum in that situation
would be unstable against the ingress of water molecules, but it is
possible in the case of fully-enclosed cavities (i.e. 'inner surface')
that are normally inaccessible to water. I don't know if anyone has
done a systematic survey of this, i.e. looking for cavities where the
density appears to be actually zero (taking into account F000 of
course), or at least significantly lower compared with the bulk
solvent density (where the assumed value of F000 wouldn't affect the
result).
Cheers
-- Ian
On 16 June 2014 07:37, James Holton <jmhol...@lbl.gov
<mailto:jmhol...@lbl.gov>> wrote:
Thank you Pavel for the clarification!
What I was really trying to point out is that a "missing atom",
occ=0.00 and occ=0.01 are not as similar as one might naiively
think. Also, if you put a ligand into a pocket and the occupancy
refines to > 0, that does not necessarily mean the ligand is
"partially occupied". If the pocket is actually filled with flat
bulk solvent, then you expect the ligand occupancy to be non-zero,
simply because something is better than nothing. However, if the
bulk solvent mask were somehow "smarter" and filled the pocket of
a, say, 60% occupied ligand with flat bulk density at 40% the
level of bulk density used far away from any atoms, then one might
actually see the occupancy of a bogus ligand refine to zero. That
is, a ligand built into a pocket that is truly "empty" (filled
with flat bulk solvent) and then occupancy refined would actually
be a "competition" between two alternative hypotheses: 1) ligand
in the pocket, 2) nothing but solvent in the pocket. If the
occupancy of the ligand refines to zero in this context, then you
can be quite confident that it didn't bind, at least not in the
given orientation.
I fully realize that the implementation of this is easier said
than done, but perhaps it would be worth the effort?
-James Holton
MAD Scientist
On 6/16/2014 3:04 PM, Pavel Afonine wrote:
Hi James,
a remark: different programs may treat occ=0 differently. In
phenix.refine (phenix.maps, etc) atoms with zero occupancy will
be ignored for bulk-solvent mask calculation, unless you ask to
do otherwise. For example, this means that if you want to
calculate a ligand OMIT map both options
- removing the ligand from PDB file;
- setting its occupancy to zero and making sure mask does not
ignore occ=0 atoms)
are a) not equivalent and b) both not good.
In first case (removing atoms from file) bulk-solvent will
flatten residual map (as you pointed out). In second case
bulk-solvent will be excluded in a very specific area, so that
residual ("green") density you see there may be either just
bulk-solvent or ligand in question or a mixture; obviously not a
very useful information! This highlights the fundamental problem
of flat bulk-solvent model the way it's currently used.
Pavel
On Sun, Jun 15, 2014 at 3:01 PM, James Holton <jmhol...@lbl.gov
<mailto:jmhol...@lbl.gov>> wrote:
The principle difference between occ=0 and omitting the atom
entirely is that occ=0 atoms exclude bulk solvent. Or at
least they do for typical operation of contemporary
refinement programs. So, by defining occ=0 you are forcing
all map voxels within ~0.6A or so of your "invisible" atom to
be vacuum. If you omit it, then the bulk solvent may "flood
in", perhaps enough to pull the fo-fc peak down below 3x
rms. How much the bulk solvent floods in depends on how
nearby atoms exclude the bulk solvent, and this, in turn,
depends on which refinement program you are using. Different
bulk solvent implementations use different radii, "shrink"
parameters, etc. In addition, bulk solvent always "bleeds" a
bit into surrounding areas because the solvent B factor is
never zero.
The real problem, I think, is that for any voxel of the map
there is ALWAYS "something there". The only question is:
what is it? Is there a 100% occupied ligand? 100% occupied
solvent? Two conformers of the ligand? Or is it some mixture
of all these? If you are asking these questions I think it
is most likely a mixture, and mixtures are hard to model.
What is worse, mixtures of a partially-occupied ligand with
bulk solvent taking up the slack is currently impossible to
model. We will have to wait for
partial-occupancy-bulk-solvent to be implemented before we
can build representations of these alternative hypotheses and
and test them with competitive occupancy refinement.
The bulk solvent is actually a very good example of something
for which we see "no evidence" in our electron density maps,
yet we model it in because 1) we know it must be there, and
2) it makes our R factors lower. What more could you want?
-James Holton
MAD Scientist
On 6/13/2014 7:45 PM, Frank von Delft wrote:
Hi all - talking about ligands, a quick question on that old
conundrum, of what to do about invisible atoms -- build them
with occ=0, or omit them?
For bits of protein, I know all the arguments; personally I
prefer omitting atoms because:
* for amino acid sidechains, their presence is implied in
the residue name.
* for whole residues, their presence is implied in the
sequence numbering
However: what about ligands? Nowhere else in the PDB file
is their presence implied - or have I missed something?
(Certainly disorder in a ligand is important information
that needs to be captured!)
Cheers
phx
