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> 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> 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
>>
>>
>>
>
>
