[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?
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
>>>>
>
> _______________________________________________
> phenixbb mailing list
> phenixbb at phenix-online.org
> http://phenix-online.org/mailman/listinfo/phenixbb
>
>
More information about the phenixbb
mailing list