If I may, I would attempt a very brief summary of this brilliant discussion.
It seems there is a general agreement on indicating, with a single parameter similar to AlphaFold's pLDDT, the reliability of the position of each atom in protein crystal structures. This would enhance the use of the PDB by non-crystallographers. This parameter could be the positional standard error, a must in small-molecule crystallography, as described by John Helliwell for protein crystal structures. This parameter depends not only on individual atomic B-factors but also on the average B-factor. However, it is not clear how to implement such a strategy for cryoEM structures. P.S. Let's celebrate the *International Women's Day.* Il giorno gio 6 mar 2025 alle ore 13:54 John R Helliwell < jrhelliw...@gmail.com> ha scritto: > 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 > > > > Dr. P. Andrew Karplus (*he, him, his)* > > Distinguished Professor Emeritus of Biochemistry and Biophysics > > NIGMS GCE4All Research Center [gce4all.oregonstate.edu] > <https://urldefense.com/v3/__http://gce4all.oregonstate.edu/__;!!PDiH4ENfjr2_Jw!ClUS-PQFSR3O42K95NZuZXa55AmbpFncgAsJv7vrQv_dV5VQZLITPkWTNbGnXm2gaqkFtfnL-rrdypo59cvHEsqiX3ww6jUY8rSxQDNBpg$> > Director > of Communications > > 2133 ALS Building > > Oregon State University > > Corvallis, OR 97331 > > ph. 541-737-3200 > > andy.karp...@oregonstate.edu > > > > *“Revealing how life works for the benefit of all!”* > > http://biochem.oregonstate.edu [biochem.oregonstate.edu] > <https://urldefense.com/v3/__http://biochem.oregonstate.edu/__;!!PDiH4ENfjr2_Jw!ClUS-PQFSR3O42K95NZuZXa55AmbpFncgAsJv7vrQv_dV5VQZLITPkWTNbGnXm2gaqkFtfnL-rrdypo59cvHEsqiX3ww6jUY8rSULgaHFw$> > / > > > > > > > > *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.] > > Thankyou for those links > > > > 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. > > > > [image: image.png] > > > > > > 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! > > > > Best wishes > > > > Matthew. > > > > > > > > > > > > > > > > > > > > > ------------------------------ > > *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 > > > > Just wanted to thank you for your remarkable contributions to this > discussion. > > These are a couple of articles that dive into the issue of unusually > large B-factors: BMC Bioinformatics 2018 19 61 > (https://doi.org/10.1186/s12859-018-2083-8 > [doi.org] > <https://urldefense.com/v3/__https://doi.org/10.1186/s12859-018-2083-8__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPIJ8Wkn7A$>) > & Zeit. Krist. 2018 234 73-77 (https://doi.org/10.1515/zkri-2018-2057 > [doi.org] > <https://urldefense.com/v3/__https://doi.org/10.1515/zkri-2018-2057__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPIIucbmag$> > ). > > Best, > > Oliviero > > > > Il giorno mar 4 mar 2025 alle ore 14:03 Frank von Delft < > 0000bcb385fe5582-dmarc-requ...@jiscmail.ac.uk> ha scritto: > > 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: > > Dear all, > > > > Fully relevant to this discussion, you might noted that a couple of years > ago, we (Vladimir Lunin and myself) argued > > > > https://journals.iucr.org/m/issues/2022/06/00/tf5001/ [journals.iucr.org] > <https://urldefense.com/v3/__https://journals.iucr.org/m/issues/2022/06/00/tf5001/__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPJrQbXHqQ$> > > > > that, when describing an atomic model, each atom should have one more > parameter, namely a local resolution with which it contributes to the map > from which it has been identified - or, in other words, with which value > its image should be calculated to reproduce the experimental map (and NOT > the density / potential itself) as a sum of atomic contributions (different > atoms may have different local resolution). > > Indicating the local resolution large (and neither B-factors large > nor occupancy small) means exactly that one cannot localize it in this > given map; again in other words, that the map from which this part of the > model was constructed had not enough information. > > > > Naturally, cif-format has no obstacle to complete the model description by > the local resolution value associated to each individual atom. Moreover, > even the old good PDB format has a space for this; positions 67-72 have > been reserved :-) > > > > Going behind the current discussion, as you perfectly know, both B-factors > and resolution cut-off blur atomic images; however they do it in a > different way (Ezra already mentioned Fourier ripples). Considering this > new parameter allows one distingushing these two effects and even to > identify (fix?) some errors occured when using the current, conventional > procedures : see, for example, Lunin et al. in Current Research in > Structural Biology (2023) : > > https://doi.org/10.1016/j.crstbi.2023.100102 [doi.org] > <https://urldefense.com/v3/__https://doi.org/10.1016/j.crstbi.2023.100102__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPJqf5yqcw$> > > > > There is a couple more of relevant articles, in Acta D and J.Appl.Cryst, > and there are works in progress. > > > > I hope, this helps... > > > > Have a nice day > > > > Sacha Urzhumtsev > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > [jiscmail.ac.uk] > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPIr_83TBw$> > > > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > [jiscmail.ac.uk] > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPIr_83TBw$> > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > [jiscmail.ac.uk] > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!PDiH4ENfjr2_Jw!GvXajeqHyc78oG7fUDHacPe5o0b-HFbzBJLEkYOApVm5CM5RsNpkyhm68SnJnELJZ8zxhNWbJBcraWaoE7qkEaKzveL3tjJenPIr_83TBw$> > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > [jiscmail.ac.uk] > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!PDiH4ENfjr2_Jw!ClUS-PQFSR3O42K95NZuZXa55AmbpFncgAsJv7vrQv_dV5VQZLITPkWTNbGnXm2gaqkFtfnL-rrdypo59cvHEsqiX3ww6jUY8rQJPt8SMg$> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
