With regards to the image that Bohdan sent and Eleanor's statements, I'm curious if the splitting of B-factors in Bohdan's image is due to the increased amount of data (which may diminish the extent of uncertainty), due to the diminished ensemble of structures within the crystal, or both. What happens to the B-factors of a structure that was derived from a 1Angstrom data set if you reduce the amount of data to 3Angstrom. In other words, you are diminishing the amount of data but not affecting the ensemble of structures that define the crystal. Perhaps I'm way off on this one....
Best wishes, Reza ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of John R Helliwell <jrhelliw...@gmail.com> Sent: 02 August 2024 11:47 AM To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: [EXTERNAL] Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era? Dear Colleagues, I think this paper from 1979 is still very interesting:- <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_280563a0&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=_Me4Xe5QZbbYN_GNBrKXdDe2jPv25n-V7XAp03Qcx-XmE9JutFEcl_X81WALv787&s=jC_Z5R86pF5k_iS5FpD1922HfoZySK0czqxWXOR8Gag&e=> Crystallographic studies of the dynamic properties of lysozyme<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_280563a0&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=_Me4Xe5QZbbYN_GNBrKXdDe2jPv25n-V7XAp03Qcx-XmE9JutFEcl_X81WALv787&s=jC_Z5R86pF5k_iS5FpD1922HfoZySK0czqxWXOR8Gag&e=> nature.com<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_280563a0&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=_Me4Xe5QZbbYN_GNBrKXdDe2jPv25n-V7XAp03Qcx-XmE9JutFEcl_X81WALv787&s=jC_Z5R86pF5k_iS5FpD1922HfoZySK0czqxWXOR8Gag&e=> [apple-touch-icon-f39cb19454.png]<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_280563a0&d=DwMFaQ&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=_Me4Xe5QZbbYN_GNBrKXdDe2jPv25n-V7XAp03Qcx-XmE9JutFEcl_X81WALv787&s=jC_Z5R86pF5k_iS5FpD1922HfoZySK0czqxWXOR8Gag&e=> Have a great weekend, John Emeritus Professor John R Helliwell DSc On 2 Aug 2024, at 16:29, Bohdan Schneider <bohdan.schnei...@gmail.com> wrote: Hello: yes, a great discussion! I second Eleanor's statement that B-factors of high resolution structures do carry a message about atom flexibility. I attach a screenshot of a figure from our paper (Schneider et al.: Local dynamics of proteins and DNA evaluated from crystallographic B factors, Acta Cryst. (2014). D70, 2413–2419) that shows clear resolution dependence of B factors at protein/protein interface for amino acids and waters. Our high resolution group of structures could not be below 1 Å as Eleanor suggests but even modest limit to 1.9 Å and then structures at 1.9-2.5 and 2.5-3.0 show the effect clearly. We looked at several other groups of atoms (backbone/side chains at the protein core, at the protein surface, DNA phosphates/bases, waters at the interfaces or bound on the protein surface) and saw the same dependence. Best, Bohdan, bs.structbio.org On 2024-08-02 13:26, Eleanor Dodson wrote: All interesting points.. (And good to see a reference to /" P.A. Machin, J.W. Campbell, M. Elder (Eds) Refinement of Protein Structures, SERC Daresbury Laboratory, Warrington, UK (1980)"/ - for those who remember, a super exciting discussion over what was feasible for refinement, and how to do it! ) My take - if a crystal diffracts to 1A we can be fairly sure of the accurate position of most of the coordinates, see other conformations for some regions, and give realistic B values to most atoms. If the crystal only diffracts to 3A then the lattice is not perfect, and there must be multiple conformations for lots of the molecule. There is not going to be sufficient experimental data to model this properly so every parameter assuming a single conformer - coordinate, B value, occupancy - is an approximation. Restraints help to some extent but they impose prior knowledge and do not glean information from the experimental data. The "trash can" should indicate the degree of uncertainty and interpreting that is a bit problematic. B values twice the overall B ?? Hmm- do NOT base too much faith in that part of the model.. As crystallographers I think maybe we need to flag this better for trusting users of the information. Omitting that region? I am not sure .. How do others model those floppy lysines? I usually make a sort of informed guess but indeed giving a single conformation is not the truth, the whole truth, and nothing but the truth.. On Fri, 2 Aug 2024 at 01:14, James Holton <jmhol...@lbl.gov> wrote: __ I submit that modern B factor restraints make them much less trashy than they were in the early days. As Pavel points out the exact strategies differ from program to program, but I don't think anybody does unrestrained B factor refinement. Not by default. Besides, all we are really doing is fitting Gaussian-shaped peaks to the "curve" of the data. These peaks have a width and a height. For example, a carbon atom with B=20 has a peak density of 1.6 e-/A^3 and a full-width-at-half-max (FWHM) of 1.4 A. That is it! That is the model density being fit. If you increase to B=80 the peak drops to 0.3 e-/A^3 and the FWHM increases to 2.6 A. At the largest B you can stuff into a PDB file (999.99), the peak height is 0.008 e-/A^3 and the "peak" is 8.45A wide. Your disordered loop, however, is probably not sampling from a symmetric Gaussian distribution like that. This is the real problem with large B factors. They can fit better than sharper B atoms, but that doesn't mean they fit well. Occupancy is easy because all it does is scale the height without affecting the width. So, an 0.5 occupancy atom model is half the height of a full-occupancy one. The width is unchanged. B factors impact both width and height because they must preserve the number of electrons in the peak. This is perhaps why they are often confusing and mysterious. We should also never forget that bulk solvent gets excluded with exactly the same radii rules from every modeled atom, regardless of B factor and occupancy. So, the "change in density" from adding or deleting an atom is a little more complicated than adding or subtracting a Gaussian peak. Nevertheless, if you want to fit peak height and width independently (like we do in pretty much every other kind of curve fitting), then you should refine occupancy and B factors at the same time. Over-fitting you say? Hardly. Polynomials are easy to over-fit, but not Gaussians. Observations/parameters is a useful guide for polynomial fits, but in general the hallmark of over-fitting is that the prediction passes exactly through all the observed points (and not the cross-validation or "Rfree" points). I have never seen a macromolecular refinement end up with Rwork = 0. Have you? At the end of the day, what we do with our models is look at their parameters and try to extract the physically meaningful reality they are trying to capture. Restraints are very helpful in preventing many types of unrealistic situations, but ultimately it is up to you to decide if the fitted model makes sense. -James Holton MAD Scientist On 7/30/2024 11:30 AM, Ian Tickle wrote: Obviously no refined parameters can ever be completely error-free, it's just that for the co-ordinates we have very accurate geometric restraints so that the relative uncertainty in the refined co-ordinates is small (but try refining co-ordinates without restraints!). For the B factors we don't have accurate estimates (if any) for their restraints so their relative uncertainty after refinement is much greater. -- Ian On Tue, Jul 30, 2024 at 6:57 PM Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com> wrote: Yes, it is and I like the definition of shared “trash bin”. It will have more physical meaning if we can separate those contributions into separate bins. Vaheh *From:* Pavel Afonine <pafon...@gmail.com <mailto:pafon...@gmail.com>> *Sent:* Tuesday, July 30, 2024 1:51 PM *To:* Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com <mailto:vaheh.oganes...@astrazeneca.com>> *Cc:* CCP4BB@jiscmail.ac.uk <mailto:CCP4BB@jiscmail.ac.uk> *Subject:* Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era? Vaheh, I think coordinates are no different from B factors, occupancies, f', or f'' in this respect. Coordinates can play their "trash bin" role by adjusting to the noise at the expense of violated geometry (bonds, angles, planes, torsions, etc.). As I mentioned in my previous email, their trash bin capacity is much smaller (but definitely not zero!) because the number and strength (confidence) of geometry restraints are much greater than those of ADP restraints. I agree that all refined parameters share this trash bin capacity, but to varying degrees. Isn't this essentially what we call the error on the refined parameter? All refined parameters have their error bars, which we have referred to as the "trash bin" in this thread. Pavel On Tue, Jul 30, 2024 at 10:09 AM Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com> wrote: Your point is taken, Pavel. However, despite resolution, you define coordinate of the atom as a geometric point with no width. Although coordinates are “refineable”, they have no capacity for “trash”. Their “trash” still goes into B-factor “trash bin”. At least this is how I see it. Thank you. *Vaheh Oganesyan, Ph.D.* *R&D **| Biologics Engineering* One Medimmune Way, Gaithersburg, MD 20878 T: 301-398-5851 _vaheh.oganes...@astrazeneca.com *From:* Pavel Afonine <pafon...@gmail.com>> *Sent:* Tuesday, July 30, 2024 11:45 AM *To:* Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com> *Cc:* CCP4BB@jiscmail.ac.uk <mailto:CCP4BB@jiscmail.ac.uk> *Subject:* Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era? From this perspective, all refinable atomic model parameters can be viewed as trash bins, with the size of these bins being proportional to the amount of prior information (restraints) imposed on these parameters. For example, coordinates have the most restraints and thus are the smallest trash bins, while B factors have the least restraints and thus are one of the largest bins. Pavel On Tue, Jul 30, 2024 at 8:25 AM Oganesyan, Vaheh <vaheh.oganes...@astrazeneca.com> wrote: Early in my Crystallography life I was postdoc with Robert Huber in Munich. We had those gatherings once a week when in very informal way we can ask and answer questions. I remember my question about B factors: how is it possible to have high resolution structure and average B-factor of 100A^2 . I think it was Robert or Albrecht Messerschmidt who told that B-factor is a “trash can” that describes not only loosely positioned atoms but also all other problems that either you created during processing, harvesting or crystal had from the beginning. *Vaheh Oganesyan, Ph.D.* *R&D **| Biologics Engineering* One Medimmune Way, Gaithersburg, MD 20878 T: 301-398-5851 _vaheh.oganes...@astrazeneca.com *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> *On Behalf Of *James Holton *Sent:* Tuesday, July 30, 2024 10:35 AM *To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> *Subject:* Re: [ccp4bb] How high a B factor is too high to assume a loop is in place, in the AlphaFold era? How high B factors can go depends on the refinement program you are using. In fact, my impression is that the division between the "let the B factors blow up" and "delete the unseen" camps is correlated to their preferred refinement program. You see, phenix.refine is relatively aggressive with B factor refinement, and will allow "missing" atoms to attain very high B factors. Refmac, on the other hand, has restraints that try to make B factor distributions look like those found in the PDB, and so tends to keep nearby B factors similar. As a result, you may get "red density" for disordered regions from refmac, inviting you to delete the offending atoms, but not from phenix, which will raise the B factor until the density fits. Then there are programs like VagaBond that don't formally have B factors, but rather let an ensemble of chains spread out in the loopy regions you are concerned about. This might be the way to go? You can also do ensemble refinement in the latest Amber. That is, you run an MD simulation of a unit cell (or more) and gradually increase structure factor restraints. This would probably result in the "fan" of loops you have in mind? -James Holton MAD Scientist On 7/28/2024 8:13 AM, Javier Gonzalez wrote: Dear CCP4bb, I'm refining the ~3A crystal structure of a big protein, largely composed of alpha helices connected by poorly-resolved loops. In the old pre-AlphaFold (AF) days I used to simply remove those loops/regions with too high B factors, because there was little to none density at 1 sigma in a 2Fo-Fc map. However, considering that the quality of a readily-computable AF model is comparable to a 3A experimental structure, and that the UniProt database is flooded with noodle-like AF models, I was considering depositing a combined model in the PDB. Once R/Rfree reach a minimum for the model truncated in poorly resolved loops, I would calculate an augmented model with AF calculated missing regions (provided they have an acceptable pLDDT value), assign them zero occupancy, and run only one cycle of refinement to calculate the formal refinement statistics. Would that be acceptable? Has anyone tried a similar approach? I'd rather do that instead of depositing a counterintuitive model with truncated regions that few people would find useful!! Thank you for your comments, Javier -- Dr. Javier M. González Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET) Universidad Nacional de Santiago del Estero (UNSE) RN9, Km 1125. Villa El Zanjón. (G4206XCP) Santiago del Estero. 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