On 3/2/2020 10:12 AM, Alexander Aleshin wrote: > Dear Dale, > You raised a very important issue that has been overly ignored by the > crystallographic community. The riding hydrogens are just a tip of an > iceberg. It is absolutely unclear even to an experienced crystallographer how > to treat poorly ordered side chains or even whole residues. As a matter of > fact, their models are "riding atoms", and consumers have no clue how much > they can trust our modeling.
Oh no! Now I've opened up this can of worms. The matter of describing completely disordered side chains has been discussed heavily on this BB, along with the advantages and shortcomings of overloading the meaning of "B factor" or "Occupancy" to describe this situation. Using one data item to describe multiple things is never a good idea, in my opinion. The move to mmCIF for model storage does open the possibility of adding new tags to uniquely describe model properties. Creating such a tag for "place-holder" side chain atoms was one of the recommendations in "Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop" (https://www.ncbi.nlm.nih.gov/pubmed/27050687). I don't know the status of the implementation of any of these recommendations. The wheels of the wwPDB grind exceedingly slowly. This is just another part of the huge problem of describing the nature of the deposited model and the origin of the information supporting all of its parts. 1) Riding Hydrogen atoms vrs free-floating and refined 2) Placeholder side chains vrs visible in density 3) Placeholder loops vrs visible in density 4) TLS anisotropic B's vrs restrained individual atom aniso vrs unrestrained individual - The options here are many and multiple types may be present in one model 5) NCS restraint/constraint - The options here are many and multiple types may be present in one model 6) Concerted alternative conformation spread over multiple residues 7) Sequence heterogeneity These are just the topics that bother me with almost every model I download. I'm sure there are plenty of other topics that don't come to mind at the moment. With the move to mmCIF we now have the opportunity to create descriptions of these model properties w/o just adding more and more REMARK cards. Until that wondrous day arrives we are stuck with trying to create models that are useful to the general community and provide minimal opportunity for confusion. We can argue as to where that line is, but shouldn't loose sight of the ultimate goal. Ethan and I disagree over the relative damage caused by the inclusion of riding hydrogen atom positions vrs the confusion that results from their absence. I think we agree strongly that all of the list items above need to be tackled by the wwPDB and are of extreme importance. I think we need a comprehensive solution, not a piecemeal, special case, for each. Dale Tronrud > Moreover, some programs (including the version of Pymol that I use), get > confused when they detect residues with multiple conformations. Like my Pymol > version fails to build cartoon elements in those areas, and it is not obvious > for a beginner how to remove the alternative conformations. I presume many > consumers just ignore such structures and pick up analogues that are > displayed without problems. > > Pymol developers, is it so difficult to report a user, when a structure is > loaded that it has residues with alternative conformations, and one of > conformers should be hidden for a correct presentation of the secondary > structure elements? > > Alex > > > > On 3/2/20, 12:57 AM, "CCP4 bulletin board on behalf of Dale Tronrud" > <CCP4BB@JISCMAIL.AC.UK on behalf of de...@daletronrud.com> wrote: > > [EXTERNAL EMAIL] > > Dear Tim, > > I am in agreement with Ethan and you that a complete description of > the restraints and constraints applied to the model should be included > in the deposition. This is currently a major failing of the wwPDB. For > hydrogen atoms we, at least, have the "Riding hydrogen atoms were added" > remark but that simple statement is inadequate to allow anyone (or > program) to reproduce what the depositor had on disk before the hydrogen > atoms were redacted. We know that shelxl and MolProbity produce > hydrogen models that differ, and that shelxl requires additional > information about the temperature of the molecule at least. > > How could someone hope to develop a better technique for generating > hydrogen atom models if the results could never be deposited and used? > > There is an additional matter of practical importance. While the two > of us share a lack of confidence in the care taken by some depositors in > the creation of hydrogen atoms, I believe the PDB customers are even > worst. If a crystallographer or microscopist should not be trusted to > add hydrogen atoms should we expect an undergrad or, maybe, a high > school student to do better? > > When someone downloads a model they expect they will be able to use > that model without performing a host of technical manipulations just to > be able to see where the depositor thought the atoms were located. We > should certainly give them enough information to understand how those > atoms were placed (and we are failing at that), but anyone should be > able to fire up Coot, load a PDB and map, and make some sense of it. > Maybe someday Coot will be able to automatically generate hydrogen > atoms, but currently the files do not contain enough information for it > to do a reasonable job. > > If hydrogen atoms are to be deleted because they can, sort of, be > recalculated, there are other aspects of the PDB file that also could be > removed. I think I could do a pretty good job of resurrecting deleted > CB atoms for any of the nineteen amino acids that contain them. Should > we just drop all CB's and add a remark saying that their locations can > be inferred from the deposited atoms? > > Dale Tronrud > > P.S. I realize that I am open to charges of inconsistency since I have > advocated not depositing an atomic model for atoms that weren't placed > by the depositor (i.e. disordered side chains). I don't believe I'm > committing this sin. I'm just saying if the depositor comes up with > locations for atoms they should be deposited. If the location of an > atom is not known it should not be deposited. I do not have a desire > for completeness for completeness' sake, just a complete listing of all > the atoms placed by the depositor. Let that high school student see our > work in all its glory! > > > On 3/1/2020 4:53 AM, Tim Gruene wrote: > > Dear Dale, > > > > your last sentence is of great importance: > > > > "leaving the (hopefully) manually inspected and curated Hydrogen atoms > in > > the deposited PDB" > > > > I believe this hope is unrealistic. Most people do probably not think or > > understand what refinement programs do about hydrogen atoms. In Refmac5 > it has > > long been an option to generate hydrogen atoms for refinement but do > not put > > them out into the PDB file. Like Ethan, I believe this is best > practice. Of > > course, in case hydrogen atoms have been curated, one may leave them in > for > > deposition. It is not useful to see all the H-atoms in Coot, and > chemists omit > > hydrogen atoms as well even for 2D drawings. > > > > @Matthew Whitley: Adding hydrogen atoms in calculated (riding) > positions > > should be rather independent of resolution of the data, since their > major role > > is in improving anti-bumping restraints, and since their major > contribution to > > the diffraction data is in the low resolution data. > > > > Best, > > Tim > > > > > > On Sunday, March 1, 2020 9:26:29 AM CET Dale Tronrud wrote: > >> Dear Ethan, > >> > >> To move away from an abstract discussion of hydrogen atoms I'd like > >> to describe a concrete example. In 2008 I deposited a model of the FMO > >> (Bacteriochlorophyll containing) protein. The ID code is 3EOJ. The > >> model was refined to a data set cut off at 1.3 A resolution using the > >> criteria of the day. I used shelxl for the final stage of refinement > >> and added riding hydrogen atoms to the mix. When I deposited the model > >> I succumb to peer pressure and removed the hydrogen atoms. > >> > >> If you look at the map calculate by the Electron Density Server you > >> will see many peaks in the Fo-Fc map indicating the missing hydrogen > >> atoms. (I have attached a screen-shot from Coot but I recommend that > >> you fire up Coot and explore the map yourself.) In my picture you can > >> see the three peaks around a methyl group. Above and to the left is > the > >> peak for the hydrogen of a CH bridging atom in the > Bacteriochlorophyll-a > >> ring. To the right and in the distance is a peak for the hydrogen of a > >> CH2 group. Not every hydrogen is represented by a positive peak, but > >> they exist throughout the map. This Fo-Fc map is useless for the > >> purpose of assessing the quality of my model, since the true residuals > >> are hidden among all these hydrogen peaks. > >> > >> A critic might say that these peaks are simply the result of the > >> model being biased toward the presence of hydrogen atoms and therefore > >> an artifact. A model refined to this data set w/o hydrogen atoms would > >> not likely show peaks indicating that hydrogen atoms need to be built. > >> > >> I would say that the map calculated from a Hydrogen-free model is > the > >> biased one. I am 99% confident in the location of most of the riding > >> hydrogen atoms and leaving them out results in a model that is > >> fantastically unlikely. The absence of peaks in an apo map is a flaw > in > >> that map. I would describe it as "vacuum bias". "Biasing" a model > >> toward reality is not a problem. > >> > >> This example shows that the current PDB is incompatible with models > >> whose Hydrogen atoms have been stripped. To get proper maps and > >> validation reports one has to either preserve the Hydrogen atoms in the > >> model, or modify all the software that uses coordinate files to add the > >> hydrogen atoms back in. That is a major programming task, which the > >> authors have, apparently, been unwilling to do. > >> > >> I will go further and disagree with you that even this is a > solution. > >> It is very difficult to add the Hydrogen atoms back into 3EOJ, and I > >> expect this difficulty is the reason software has not been written that > >> successfully does it. > >> > >> There are two major problems to be overcome in 3EOJ. shelxl has an > >> option to twirl the methyl groups and select the torsion angle with the > >> best fit to the map. The hydrogen atoms in the pictured methyl group > >> weren't built as staggered -- All values for the torsion angle were > >> tested and it happens that the best fit placed them in a staggered > >> conformation. That is a much more interesting result. There are other > >> methyl groups around the edges of the Bchl-a molecules that are crowded > >> and the methyl groups are observed to have torsion angles that are not > >> standard for riding Hydrogen atoms. The neighboring methyl groups > avoid > >> H-H bumps by twisting and that twist can be detected by shelxl in the > >> 1.3 A data. > >> > >> The second problem is the matter of Histidine residues. There are > >> two Nitrogen atoms in the side chain. A hydrogen atom could be on > >> either one, and sometimes both have hydrogens. A very clever program > >> could work out from the hydrogen bonding pattern the most likely > >> placement, but I've not seen any program that is very good with > hydrogen > >> bonding networks. Worst still, I've often seen programs place the > >> hydrogen atom *between* the Nitrogen and Magnesium atoms of a Histidine > >> ligand to a Bacteriochlorophyll a. This mistake will certainly lead to > >> very bad geometry! > >> > >> Until an hydrogenation program is written that can handle all > >> ligands, all hydrogen bonding networks (even overlapping partially > >> occupied ones), and use the imaging data to place the Hydrogen atoms > >> that have one or two dimensional mobility, I don't see an alternative > to > >> leaving the (hopefully) manually inspected and curated Hydrogen atoms > in > >> the deposited PDB. > >> > >> Dale Tronrud > >> > >> P.S. > >> I'm hoping to find the time to use the new versioning capability of > >> the wwPDB to put my Hydrogen atoms back in 3EOJ. > >> > >> On 2/28/2020 10:34 PM, Ethan A Merritt wrote: > >>> Matthew: > >>> > >>> I think your nice summary leaves out an important point that has not > been > >>> explicitly mentioned. That is the question of whether depositing > >>> hydrogens > >>> actually adds information to the model. I submit that for a typical > >>> protein > >>> refinement it does not. The model is adequately described by saying > >>> "hydrogens were added in their riding positions". This, together with > >>> knowledge of the refinement program used, is sufficient to reconstruct > >>> the full model. > >>> > >>> This is an example of a recurring concern of mine that model > validation > >>> should include consideration of whether the model is overly complex. > >>> Unless you have an abundance of data (which admittedly your 1.0Å case > >>> might) there are insufficient observations to refine 3 positional > >>> parameters for each hydrogen as if they were free variables. We > >>> typically bypass this by instead using the riding hydrogen model, > which > >>> adds effectively a single on/off parameter for the entire mode (plus a > >>> small number of implicit parameters that describe the ideal riding > >>> geometry, but those are > >>> normally taken as a priori knowledge rather than free variables). > >>> > >>> So I find deposition of hydrogens for a typical resolution structure > to > >>> be more misleading than useful. The correct, parsimonious, > description is > >>> the one-line statement that a riding hydrogen model was used. > >>> > >>> It is tangential to your question, but I hold the same view about > >>> depositing ANISOU records for a structure when the source of the > >>> anisotropy is solely a TLS model, either with or without individual > Biso > >>> contributions. The parsimonious description is to give the TLS > parameters > >>> and the Biso component, if any. These can be expanded to regenerate > >>> per-atom > >>> ANISOU parameters if desired by a downstream program. > >>> If you deposit ANISOU records it implies that the Uij terms they > describe > >>> are free variables, but they are not. (or anyway IMHO they should > not be, > >>> although PHENIX can violate this stricture). > >>> > >>> My view is that for a typical structure (i.e. worse than say 1Å > resolution > >>> data) depositing hydrogen positions and ANISOU records at best does no > >>> harm. Unfortunately it implies a statistically unjustifiable model > >>> treatment. The justifiable model is adequately described by the small > >>> number of parameters in the header records; the hydrogen coordinates > and > >>> ANISOU parameters are redundant dross. > >>> > >>> I fully understand that your original question was driven by cases > where > >>> you do have very high resolution data and so the statistical > justification > >>> of refining individual hydrogens or anisotropic ADPs enters a > different > >>> realm.> > >>> Ethan > >>> > >>> On Friday, 28 February 2020 20:22:17 PST Whitley, Matthew J wrote: > >>>> Dear all, > >>>> > >>>> I want to thank everyone who responded to my query about whether or > not > >>>> to > >>>> include hydrogens in PDB depositions when they were explicitly > included > >>>> in > >>>> the model during refinement. In addition to the replies posted to > this > >>>> bulletin board, I received numerous replies sent directly to my email > >>>> address. > >>>> > >>>> To clarify one more time for casual readers so that we are all on the > >>>> same > >>>> page: because these two structures happen to be at high resolution > (1.0 > >>>> and > >>>> 1.2 Å, respectively), I decided to include explicit hydrogens in the > >>>> model > >>>> for refinement, as recommended by the documentation for both Phenix > and > >>>> Buster, which I used for these refinements. For the Phenix > refinements, > >>>> hydrogens were added by phenix.ready_set, whereas for the Buster > >>>> refinements the hydrogenate tool was used. My understanding is that > both > >>>> of these eventually call the reduce tool from MolProbity. > >>>> Unsurprisingly, > >>>> the presence of hydrogens on the model led to both better model > geometry > >>>> and lower R-factors, although at these resolutions there is no > observable > >>>> density for the vast majority of the H-atoms in any of the refined > maps. > >>>> > >>>> Because the presence of the hydrogens improved the model, I have > decided > >>>> to > >>>> leave the hydrogens at their refined positions for deposition. > >>>> > >>>> I do want to point out one thing for readers interested in this > topic: > >>>> based on all the replies I received, there are a number of differing > >>>> opinions (and therefore different practices) as to whether hydrogens > >>>> should be included in deposited structures. The expressed opinions > >>>> ranged from the ethical (if the hydrogens were there for refinement, > >>>> then it’s only fair that they be present in the deposited structure > so > >>>> that downstream users know what went into generating the reported > >>>> statistics) to the practical (if the paper’s conclusions don’t rely > on > >>>> any arguments based on hydrogen atom positions, then there’s no > >>>> compelling reason for them to be there; include them or don’t, it > >>>> doesn’t matter.) Because opinions seem to vary, perhaps it would be > >>>> worthwhile for the PDB to issue some guidance on the matter for the > >>>> future. > >>>> > >>>> Have a nice weekend, everyone. > >>>> > >>>> Matthew > >>>> > >>>> --- > >>>> Matthew J. 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