[phenixbb] TLS and ligand question

[Andy Torelli]

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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