[phenixbb] Using LigandFit to identify unknown density

Maia Cherney chern at ualberta.ca
Wed Jan 27 17:01:14 PST 2010

Thanks, Pavel,

now I got it.


Pavel Afonine wrote:
> 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.
> Pavel.
> 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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