[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.
Maia
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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