[phenixbb] Using LigandFit to identify unknown density
PAfonine at lbl.gov
Wed Jan 27 14:51:23 PST 2010
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?
******* 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.
> 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.
>> 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.
>>> ----- 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 (
>>> which I have become a big fan of.
>>> Kendall Nettles
>>> On 11/24/09 3:07 PM, "chern at ualberta.ca" <chern at ualberta.ca> wrote:
>>> I am wondering what is the criteria for occupancy refinement of
>>> ligands. I noticed that R factors change very little, but the
>>> 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?
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