[phenixbb] Using LigandFit to identify unknown density

Pavel Afonine PAfonine at lbl.gov
Wed Jan 27 15:34:27 PST 2010

Hi Maia,

phenix.refine refines occupancies during occupancy refinement, it 
refines B-factors during B-factor refinement and it refines coordinates 
during coordinate refinement. The B-factor restraints are applied at 
B-factor refinement step. phenix.refine iterates these steps as many 
times as large the main.number_of_macro_cycles parameter is (3, by 
default). Obviously, no B-factor are restraints applied if you refine 
occupancies only.

Yes, what Peter mentioned actually happens during refinement (if 
B-factor refinement is enabled). That's what the B-factor restraints do 
in general.


On 1/27/10 3:28 PM, Maia Cherney wrote:
> 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?
> Maia
> 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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