[phenixbb] TLS and ligand question
att29 at cornell.edu
Wed Nov 25 13:13:52 PST 2009
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 ;).
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:
>> 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,
>> 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.
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