Dear Matthew,I suggest that if you are able to make the placements you mention that you do so, then calculate for the non covalent pairs of atoms that might be interacting (H bonds or van der Waals) coordinate errors and proceed as cautiously as those uncertainty estimates on distances suggest your narrative should be. The Online DPI webserver offers one such https://journals.iucr.org/paper?vg5015 a way forward. Or alternatively multiple workflows to look at the refined models based on the same diffraction dataset give you coordinate (and B factor) variances too. Also make your raw diffraction data available as well. Obviously I don’t know all the details, but as a referee who scrutinises article, with the to be released PBB files, and the raw data if needs be, it sounds that I would be satisfied with yours. A further check would be if you have replicates you could document those as well for the reader, attaching those data to the article as supplementary evidence. Best wishes, John Emeritus Professor John R Helliwell DSc
On 6 Mar 2025, at 11:20, Matthew Snee <matthew.sne...@manchester.ac.uk> wrote:
I'm sorry to everyone for still banging on about this, I'm sure everyone is well and truly bored with it, but I consider my structure to be useful/interesting (at least to me) despite its strangeness and limitations
And I really don't want to end up in someone's "what not to do" slideshow at some conference or other!
I'm trying to digest the literature about high-B factors, but it mostly pertains to examples where researchers are building unobserved features into otherwhise strong structures, the assertion being that they have no exerimental evidence (imprecise or otherwise)
to inform the position of these atoms.
I believe mine is an edge case where the Scaling B factor and the refinement B factors are (inappropriately but possibly unavoidably) accounting for the large range of flexibility.
The Wilson B factor therefore reaches over 100, and a whole mobile domain (which I estimate to have a local res more like 5 Angstrom) has protein B factors around 200!
I have of course verified that this domain is indeed present, and although its challenging you can see features that that werent in the search model (PTMS etc..) even prior to refinement.
I would argue that none of the B factors/ADPs are good representations of the true thermal motion/vibration of the atoms, even the ones that are close or significantly below the wilson B value.
The detail is however perfectly adequate to model sidechains and other features for most of the structure, and there are useful distinctions between the real structure and the prediction (AF3).
Pavel had some useful advice about not using B factors to describe things that should be described by occupancy (even if you end up having less than 100% occupancy for single modeled conformer of a covalent feature which is "present" more or less 100% of the
time like the glycan).
The problem is that this principle would apply to whole domains, and actually the whole structure if what I think is correct.
In a crystal with a great degree of flexibility (and a wide range in relative flexibility), you could argue that the presented conformation definitely has an occupancy of less than 1.0 I guess?
I wouldn't be upset at the assertion that the B values are "wrong" and this structure should be excluded from any work studying B factors (or perhaps the opposite to improve the way disorder is accounted for), but its the claim that the atomic coordinates have
been modelled carelessly that I would like to avoid!
Obviously, I will discuss this openly in the publication, and only rely on features that are unambiguous for my conclusions, but it would be good to know peoples thoughts on what they would do or expect to see.
Best
Matthew.
From: Karplus, Andy <andy.karp...@oregonstate.edu>
Sent: 06 March 2025 01:02
To: Matthew Snee <matthew.sne...@manchester.ac.uk>; CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] IDS in PDB
Hi Matthew.
Your post reminds me of some work my student did earlier related to the question of what to consider as “too high a B-factor.” Back in 2003 a referee raised concerns about the “way too high” B-factors of
a 2.6 Å resolution structure we had determined, because the average B-factor of the structure was about 85 Å2. Even though the density for the modeled parts of the structure was quite clear that was a red flag for the reviewer.
In response we provided a variety of evidences to validate the structure, and then also briefly explored whether the structure having an average B-factor much higher than was then considered reasonable for
a structure at 2.6 Å resolution could be at least in part related to our use of a synchrotron source for the data collection. Our thought was that the greater X-ray intensity could lead to data with notable signal out to 2.6 Å resolution for a crystal that
perhaps in a lab setting might have only yielded data with notable signal out to a much lower resolution.
To test this idea, we looked at PDB structures and generated a plot comparing the average B-factor for structures determined using a lab source versus a synchrotron source. There was a difference, with distribution
of structures based on synchrotron radiation notably shifted to higher average B-factors (extending up to ~100 Å2
compared with ~70 Å2). And crucial to remember when looking at this plot is that for many (perhaps most) structures determined at say 2.5 – 3.0 Å resolution, the resolution limit at which the structure was determined need not reflect the limit
to which useful data could have been collected. For instance, for a protein crystal with a size and level of internal order that would allow for meaningful data to be collected out to 1 Å resolution, researchers looking at a series of ligand bound structures
may choose to collect them all at 2.5 Å resolution, knowing that that is much faster and provides enough information to answer the questions they are asking; and these structure would give very low refined B-factors compared with a structure from a large crystal
with a level of internal order that truly leads to 2.5 Å as the limit of resolution to which useful data can be collected under the best circumstances.
The analysis I’m referring to is in Figure 2D of the paper at doi:10.1016/S0022-2836(03)00347-4 .
HTH, Andy
From:
CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Matthew Snee <matthew.sne...@manchester.ac.uk>
Date: Wednesday, March 5, 2025 at 11:19 AM
To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] IDS in PDB
[This email originated from outside of OSU. Use caution with links and attachments.]
One of those papers seems to basically say that atoms that accumulate stratospheric B factors are "speculative", but Im working on an example with a Wilson B factor of over 100 where some of
the weaker features have enormous B values.
The crystal is very atypical, very high solvent, and a very wide range of flexibility where whole domains can move almost freely in solvent channels, but are still observed.
The features that have obtained super high B factors are the glycans. The general shape of the core Glycan can be seen clearly, but the conformation is not constrained by any contacts, so the
density is extremely diffuse.
The "answer" is obviously that there is an ensemble of conformations (for these features and also a whole domain), which would exist at low occupancy but would also accrue a lower B factor,
but I think it is more appropriate to model a single favourable conformer that fits the density.
This is certainly the first X-ray structure that I ever worked on that was quite like this, so id like to hear peoples opinions on how I should handle it. The values out of context would certainly
raise eyebrows!
I work quite a bit on EM and it appears really similar to some EM examples with resolution ranges between 2.8-6A.
I am certain that the features with the very high values are much more than speculative, and the model is more accurate when complete, but the B factors are certainly not describing the relationship
between the model and the data in a useful way!
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Italo
Carugo Oliviero <olivieroitalo.car...@unipv.it>
Sent: 05 March 2025 16:21
To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] IDS in PDB
Interesting...
Has this got onto the radar (or critical path) of the PDB's mmCIF working group (or whatever it's called?)
I'm assuming that's where this would go to next, if the downstream developers are ever going to take it seriously.
Frank
On 04/03/2025 12:21, Alexandre Ourjoumtsev wrote:
Fully relevant to this discussion, you might noted that a couple of years ago, we (Vladimir Lunin and myself) argued
that, when describing an atomic model, each atom should have one more parameter, namely a local resolution with which it contributes to the map from which it
has been identified - or, in other words, with which value its image should be calculated to reproduce the experimental map (and NOT the density / potential itself) as a sum of atomic contributions (different atoms may have different local resolution).
Indicating the local resolution large (and neither B-factors large nor occupancy small) means exactly that one cannot localize it in this given map; again
in other words, that the map from which this part of the model was constructed had not enough information.
Naturally, cif-format has no obstacle to complete the model description by the local resolution value associated to each individual atom. Moreover, even the old
good PDB format has a space for this; positions 67-72 have been reserved :-)
Going behind the current discussion, as you perfectly know, both B-factors and resolution cut-off blur atomic images; however they do it in a different way (Ezra
already mentioned Fourier ripples). Considering this new parameter allows one distingushing these two effects and even to identify (fix?) some errors occured when using the current, conventional procedures : see, for example, Lunin et al. in Current Research
in Structural Biology (2023) :
There is a couple more of relevant articles, in Acta D and J.Appl.Cryst, and there are works in progress.
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