[phenixbb] Using LigandFit to identify unknown density

Maia Cherney chern at ualberta.ca
Wed Jan 27 15:28:28 PST 2010

Hi Pavel, Peter,

Thank you for your reply. My question is if the phenix.refine actually 
uses the B-factor restraints in the occupancy refinement. I did not give 
any restraints, so it should happen automatically? I like the idea that 
Peter mentioned that the restraints should make B -factors similar to 
surrounding molecules. Again, my question is does phenix.refine actually 
uses this approach?


Pavel Afonine wrote:
> 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.
> Pavel.
> 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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