[phenixbb] Using LigandFit to identify unknown density

Pavel Afonine PAfonine at lbl.gov
Wed Jan 27 14:51:23 PST 2010

Hi Maia,

first, I agree with Peter - the B-factor restraints should help, indeed.

Second, I think we discussed this subject already on November 25, 2009:

Subject: Re: [phenixbb] occupancy refinement
Date: 11/25/09 7:38 AM

and I believe I didn't change my mind about it since that. I'm appending 
that email conversation to the bottom of this email.

Overall, if you get good 2mFo-DFc map and clear residual mFo-DFc map, 
and ligand's B-factors are similar or slightly larger than those of 
surrounding atoms, and refined occupancy looks reasonable, then I think 
you are fine.


On 1/27/10 2:05 PM, Maia Cherney wrote:
> Hi Pavel,
> I have six ligands at partial occupacies in my structure. Simultaneous 
> refinement of occupancy and B factors in phenix gives a value of 0.7 
> for the ligand occupancy that looks reasonable.
> How does phenix can perform such a refinement given the occupancies 
> and B factors are highly correlated? Indeed, you can increase/decrease 
> the ligand occupancies while simultaneously increacing/decreasing 
> their B factors without changing the R factor value. What criteria 
> does phenix use in such a refinement if R factor does not tell much?
> Maia 

******* COPY (11/25/09)************

On 11/25/09 7:38 AM, Maia Cherney wrote:
> Hi Pavel,
> It looks like all different refined occupancies starting from different 
> initial occupancies converged to the same number upon going through very 
> many cycles of refinement.
> Maia
> Pavel Afonine wrote:
>> Hi Maia,
>> the atom parameters, such as occupancy, B-factor and even position are 
>> interdependent in some sense. That is, if you have somewhat incorrect 
>> occupancy, that B-factor refinement may compensate for it; if you 
>> misplaced an atom the refinement of its occupancy or/and B-factor will 
>> compensate for this. Note in all the above cases the 2mFo-DFc and 
>> mFo-DFc maps will appear almost identical, as well as R-factors.
>> So, I think your goal of finding a "true" occupancy is hardly achievable.
>> Although, I think you can approach it by doing very many refinements 
>> (say, several hundreds) (where you refine occupancies, B-factors and 
>> coordinates) each refinement starting with different occupancy and 
>> B-factor values, and make sure that each refinement converges. Then 
>> select a subset of refined structures with similar and low R-factors 
>> (discard those cases where refinement got stuck for whatever reason 
>> and R-factors are higher) (and probably similar looking 2mFo-DFc and 
>> mFo-DFc maps in the region of interest). Then see where the refined 
>> occupancies and B-factors are clustering, and the averaged values will 
>> probably give you an approximate values for occupancy and B. I did not 
>> try this myself but always wanted to.
>> If you have a structure consisting of 9 carbons and one gold atom, 
>> then I would expect that the "second digit" in gold's occupancy would 
>> matter. However, if we speak about dozen of ligand atoms (which are 
>> probably a combination of C,N,O) out of a few thousands of atoms of 
>> the whole structure, then I would not expect the "second digit" to be 
>> visibly important.
>> Pavel.
>> On 11/24/09 8:08 PM, chern wrote:
>>> Thank you Kendall and Pavel for your responces.
>>> I really want to determine the occupancy of my ligand. I saw one 
>>> suggestion to try different refinements with different occupancies 
>>> and compare the B-factors.
>>> The occupancy with a B-factor that is at the level with the average 
>>> protein B-factors, is a "true" occupancy.
>>> I also noticed the dependence of the ligand occupancy on the initial 
>>> occupancy. I saw the difference of 10 to 15%, that is why I am 
>>> wondering if the second digit after the decimal point makes any sence.
>>> Maia
>>>     ----- Original Message -----
>>>     *From:* Kendall Nettles <mailto:knettles at scripps.edu>
>>>     *To:* PHENIX user mailing list <mailto:phenixbb at phenix-online.org>
>>>     *Sent:* Tuesday, November 24, 2009 8:22 PM
>>>     *Subject:* Re: [phenixbb] occupancy refinement
>>>     Hi Maia,
>>>     I think the criteria for occupancy refinement of ligands is
>>>     similar to a decision to add an alt conformation for an amino
>>>     acid. I don’t refine occupancy of a ligand unless the difference
>>>     map indicates that we have to. Sometimes part of the igand may be
>>>     conformationally mobile and show poor density, but I personally
>>>     don’t think this justifies occupancy refinement without evidence
>>>     from the difference map. I agree with Pavel that you shouldn’t
>>>     expect much change in overall statistics, unless the ligand has
>>>     very low occupancy., or you have a very small protein. We
>>>     typically see 0.5-1% difference in R factors from refining with
>>>     ligand versus without for nuclear receptor igand binding domains
>>>     of about 250 amino acids, and we see very small differences from
>>>     occupancy refinement of the ligands.
>>>     Regarding the error, I have noticed differences of 10% percent
>>>     occupancy depending on what you set the starting occupancy before
>>>     refinement. That is, if the starting occupancy starts at 1, you
>>>     might end up with 50%, but if you start it at 0.01, you might get
>>>     40%. I don’t have the expertise to explain why this is, but I
>>>     also don’t think it is necessarily important. I think it is more
>>>     important to convince yourself that the ligand binds how you
>>>     think it does. With steroid receptors, the ligand is usually
>>>     planer, and tethered by hydrogen bonds on two ends. That leaves
>>>     us with with four possible poses, so if in doubt, we will dock in
>>>     the ligand in all of the four orientations and refine. So far, we
>>>     have had only one of several dozen structures where the ligand
>>>     orientation was not obvious after this procedure. I worry about a
>>>     letter to the editor suggesting that the electron density for the
>>>     ligand doesn’t support the conclusions of the paper, not whether
>>>     the occupancy is 40% versus 50%.
>>>     You might also want to consider looking at several maps, such as
>>>     the simple or simulated annealing composite omit maps. These can
>>>     be noisy, so also try the kicked maps (
>>>     http://www.phenix-online.org/pipermail/phenixbb/2009-September/002573.html),
>>>     <http://www.phenix-online.org/pipermail/phenixbb/2009-September/002573.html%29,>
>>>     which I have become a big fan of.
>>>     Regards,
>>>     Kendall Nettles
>>>     On 11/24/09 3:07 PM, "chern at ualberta.ca" <chern at ualberta.ca> wrote:
>>>         Hi,
>>>         I am wondering what is the criteria for occupancy refinement of
>>>         ligands. I noticed that R factors change very little, but the
>>>         ligand
>>>         B-factors change significantly . On the other hand, the
>>>         occupancy is
>>>         refined to the second digit after the decimal point. How can
>>>         I find
>>>         out the error for the refined occupancy of ligands?
>>>         Maia

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