[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.
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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