Hi Maia, I think the B values of your ligand have restraints on them encouraging them to be similar to surrounding atoms. This additional info reduces the dependence between occupancy and B value. HTH P 2010/1/27 Maia Cherney :

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

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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:[email protected] *To:* PHENIX user mailing list mailto:[email protected] *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%2...

which I have become a big fan of.

Regards, Kendall Nettles

On 11/24/09 3:07 PM, "[email protected]" 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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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:[email protected] *To:* PHENIX user mailing list mailto:[email protected] *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%2...

which I have become a big fan of.

Regards, Kendall Nettles

On 11/24/09 3:07 PM, "[email protected]" 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 [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

_______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb

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