[phenixbb] TLS and ligand question

[Andy Torelli]

Dale,

	Thank you for taking the time to respond, your comments were very 
helpful.  I plan to try the group-occupancy refinement as both you and 
Pavel have suggested (and be thankful that my analysis does not depend 
on an accurate measure of occupancy ;).

Best Regards,
-Andy Torelli

On 11/25/2009 3:42 PM, Dale Tronrud wrote:
>   The statement that occupancy and B factor are correlated means that
> you can increase or decrease the occupancy while simultaneously
> increasing or decreasing the B factor w/o changing the R value much.
> This means that the R value can't tell you much about the correct
> settings.  Since the R value is just a measure of the overall r.m.s.
> of the difference map (basically), the difference map isn't going to
> help much either.
> 
>    If you have high enough resolution to see features in the map when
> you change occupancy and B factor then those combinations are ruled
> out, but refinement will not generate those combinations because the
> goal of refinement is to flatten difference maps.
> 
>    You can't get around the fact that at medium resolutions this
> correlation exits and will cause your occupancy and B factor parameters
> to have large error bars.  Forcing the occupancy to be equal to one
> can make you feel better, but from you description it is clear that
> that model is incorrect.  You are trying to decide between an
> incorrect model that has the illusion of precision and a model that
> optimally fits your data but is imprecise.
> 
>    If I believe a ligand is only partially occupied I'll perform
> the group occupancy refinement and use the comparison of the average
> B factor to the neighboring B factors as a validation, but note in
> any mention of the final occupancy its high uncertainty.  If some
> conclusion of you paper depends on the occupancy being 50% and not
> 60% you had better try some other technique - 2 Angstrom crystallography
> is not going to do the trick for you.
> 
>    Remember, when your compound is not present it is likely that
> water molecules will be.  The strongest features in your density are
> likely to be places that switch between your compound and water.
> If you have an apo crystal model you can find these waters easily,
> otherwise you will have to deduce their locations from these strong
> regions of density.
> 
> Dale Tronrud
> 
> Andy Torelli wrote:
>> Pavel,
>>
>> 	Thanks for the suggestions.  I will try group occupancy refinement as 
>> you suggest.  Consequently, I "know" that at intermediate resolution the 
>> B-factor and occupancy are coupled and my understanding was that it 
>> isn't appropriate to refine both.  However, I haven't read anything that 
>> rigorously describes when it is appropriate to do so (i.e. at what 
>> resolutions or other characteristics of the data).  How can one tell 
>> whether the result of refining both B-factor and occupancy 
>> simultaneously (one occupancy per group in the case of ligands) achieves 
>> a reasonable result?  Obviously you'd expect potentially to see lower 
>> R/Rfree, more uniform B-factors and better electron density (i.e. no or 
>> reduced difference electron density).  Conversely, what would indicate 
>> to you that it is inappropriate to refine both together?
>>
>> 	Also, I didn't mention that I also tried to set all the isotropic 
>> B-factors to a uniform value before running TLS (recommended in Martyn 
>> Winn's manual), but that didn't help.
>>
>> Thanks for your advice,
>> -Andy
>>
>> On 11/25/2009 1:30 PM, Pavel Afonine wrote:
>>> Hi Andy,
>>>
>>>> 	I've been experimenting with using TLS to refine my 2.4 angstrom 
>>>> resolution structure.  There are 2 protomers in the ASU, and each has a 
>>>> metal cluster and a small-molecule ligand.  The model has a relatively 
>>>> high average B-factor for protein (~65) atoms and I also suspect that 
>>>> both the cluster and the ligand have partial occupancy.  At this 
>>>> resolution however, I've modeled both groups with 100% occupancy and the 
>>>> average B-factors for the atoms of these groups are expectedly higher, 
>>>> ~100 after individual_sites and individual_adp refinement (i.e. no TLS).
>>>>   
>>> Still, I would try refining group occupancy factor for ligands (one 
>>> refinable occupancy per whole ligand), and before doing so I would 
>>> re-set ligand's B-factors to an average value.
>>>
>>>> 	My problem arises when I try to use TLS.  I determined the boundaries 
>>>> for the TLS groups using the TLSMD server, which identified 3 groups per 
>>>> protomer (roughly coincide with domain boundaries).  After including TLS 
>>>> refinement, the R and R-free both improve by 1-2%, but the B-factors for 
>>>> the clusters and ligands end up very high.  
>>> As far as I know there is still no consensus about if one needs to 
>>> include water, ions or small ligands into TLS groups. I did a few 
>>> experiments in the past and at that point I found that not including 
>>> them into TLS groups worked better. But I was using really a few test 
>>> structures so this is not conclusive.
>>> I would not include them into TLS groups, and try refining group occupancy.
>>>
>>>> Metal atoms in the cluster 
>>>> have B-factors >350 and the ligand atoms have B-factors >120.  The 
>>>> average B-factor for protein atoms is slightly higher too (~70).  Also, 
>>>> the gap between R and Rfree is larger than I'd expect (19% R and 26% 
>>>> Rfree), although this isn't unheard of and more importantly, the values 
>>>> have fully converged yet.
>>>>   
>>> Try optimizing weights: "optimize_wxc=true optimize_wxu=true", it may help.
>>>
>>> Pavel.
>>>
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