Re: [ccp4bb] chloride or water
After reading this exchange, I think at the core of the dispute is the question what a structure model really is supposed to represent (a), and how to annotate/describe it (b). ad (a) In general, and forgive me for not disclosing all caveats and fine tune (I leave this to GB), we are interested in the posterior likelihood (model likelihood). The two terms to consider (yes, I know, I am omitting any normalization necessary for hypothesis testing etc) this model likelihood would be proportional to the product of an evidence term (data likelihood) and an independent prior knowledge term. Imho the expressed opinions diverge primarily in the relative significance of the terms or normalization of the probabilities. The evidence purists (and it seems that computationalists often mistake this for arrogance of the crystallographers) argue that if I can’t see/recognize it in ED or support it otherwise by direct experimental evidence, leave it out of the model (after all, X-ray structure models are supposed to be based on experimental evidence). On the other hand, from prior knowledge (admittedly extracted from polluted data bases like the PDB and that is not meant as an insult but a statement of fact) we do know something about what reasonably could be expected and could use it to the full extent of its statistical support. Both extremes are of course justifiable, but in practice not separable. E.g. we use riding hydrogens without giving it a second thought that we do not see them in (macro X-ray) ED, and they do improve models. On the other hand, we still put side chain atoms we do not ‘see’ in specific positions and hope that the B-factors increase to a point where the absence of any meaningful scattering contributions does not ruin our Holy R. That specific position is perhaps closer to ‘wild speculation’ than the probability that a chloride atom exists in that specific case. (I do argue that in the above case a set of conformations with occupancies of rotamers corresponding to their population in the torsion angle landscape (or in the polluted databases) – the prior – under consideration where they cannot be – the rest of the model as obtained from evidence – would be a possible description). The final weighting one could apply might be a less tangible factor – how badly does it matter? If a ligand in a specific pose is modelled and intended for the use of drug discovery, I’d say the claim is extraordinarily strong, and the model likelihood (both terms) better be convincing. In the less earth shaking blob case, considering priors and the mentioned restrictions of low resolution etc, I can accept a low but not unreasonable probability (- such apparent evasiveness being a dead giveaway of a mental Bayes factor calculation instead of adherence to an artificial significance level; frequentists please feel free to flame me) for Cl as the most probable in the Cl/water/empty model competition (not that any of the models are overly convincing, however, compatible with the low drama factor of that decision). ad (b) having said this, how to express such probabilistic considerations in the current atomic PDB model format, is an unresolved issue. I think the whole idea of the single static atomic model sooner or later will fall. It is already a mess because much information about the model is hidden for example in remarks like TLS groups (btw, one of the most abused and ad-hoc applied means in the hope of reducing Holy R instead of reflecting what these groups actually mean). But this is besides the original point and becoming free floating… I am not calling for making peace here, rather argue that the seemingly insignificant issue of a single Cl ion in one of 100k structure models can lead to productive reflection about meaning and improvement of model description. Sorry for offending those in need for cozy comfort closing quotes. The answer is, as always, 42. HTC, BR (Happy To Confuse) From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Mittwoch, 21. Januar 2015 19:18 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water I reiterate that assigning a chloride is not “wild speculation” or “just making something up” in light of what we know about the situation. I see your point about not knowing that it’s a chloride, but I think you would agree that it is certainly more likely a chloride than map-noise, and perhaps more likely than water as well. Would you agree that chloride is the best guess, at least? What are the options for that blob, and what is the probability of each? I think you want to make sure people don’t get misled by it, which is a good point and a noble aspiration. I would argue that “not choosing” is here, as everywhere else, indeed choosing. And if you choose nothing here, you are almost certainly wrong, given
Re: [ccp4bb] chloride or water
At the risk of derailing the discussion, can it be that the blob is actually an accumulation of many Fourier ripples? (on top of bulk solvent, I guess). The “chloride” seems to be about 3.5Å away from a lot of atoms, with nothing closer. This is mostly based on intuition and the fact that in my experience any almost spherical cavity or any almost cylindrical crevice has a blob of difference density inside, which often proves to be very difficult to model. I have no hard data to back this up. Cheers, Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dkmailto:josea.cuesta.se...@carlsberglab.dk From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thursday, January 22, 2015 10:26 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water After reading this exchange, I think at the core of the dispute is the question what a structure model really is supposed to represent (a), and how to annotate/describe it (b). ad (a) In general, and forgive me for not disclosing all caveats and fine tune (I leave this to GB), we are interested in the posterior likelihood (model likelihood). The two terms to consider (yes, I know, I am omitting any normalization necessary for hypothesis testing etc) this model likelihood would be proportional to the product of an evidence term (data likelihood) and an independent prior knowledge term. Imho the expressed opinions diverge primarily in the relative significance of the terms or normalization of the probabilities. The evidence purists (and it seems that computationalists often mistake this for arrogance of the crystallographers) argue that if I can’t see/recognize it in ED or support it otherwise by direct experimental evidence, leave it out of the model (after all, X-ray structure models are supposed to be based on experimental evidence). On the other hand, from prior knowledge (admittedly extracted from polluted data bases like the PDB and that is not meant as an insult but a statement of fact) we do know something about what reasonably could be expected and could use it to the full extent of its statistical support. Both extremes are of course justifiable, but in practice not separable. E.g. we use riding hydrogens without giving it a second thought that we do not see them in (macro X-ray) ED, and they do improve models. On the other hand, we still put side chain atoms we do not ‘see’ in specific positions and hope that the B-factors increase to a point where the absence of any meaningful scattering contributions does not ruin our Holy R. That specific position is perhaps closer to ‘wild speculation’ than the probability that a chloride atom exists in that specific case. (I do argue that in the above case a set of conformations with occupancies of rotamers corresponding to their population in the torsion angle landscape (or in the polluted databases) – the prior – under consideration where they cannot be – the rest of the model as obtained from evidence – would be a possible description). The final weighting one could apply might be a less tangible factor – how badly does it matter? If a ligand in a specific pose is modelled and intended for the use of drug discovery, I’d say the claim is extraordinarily strong, and the model likelihood (both terms) better be convincing. In the less earth shaking blob case, considering priors and the mentioned restrictions of low resolution etc, I can accept a low but not unreasonable probability (- such apparent evasiveness being a dead giveaway of a mental Bayes factor calculation instead of adherence to an artificial significance level; frequentists please feel free to flame me) for Cl as the most probable in the Cl/water/empty model competition (not that any of the models are overly convincing, however, compatible with the low drama factor of that decision). ad (b) having said this, how to express such probabilistic considerations in the current atomic PDB model format, is an unresolved issue. I think the whole idea of the single static atomic model sooner or later will fall. It is already a mess because much information about the model is hidden for example in remarks like TLS groups (btw, one of the most abused and ad-hoc applied means in the hope of reducing Holy R instead of reflecting what these groups actually mean). But this is besides the original point and becoming free floating… I am not calling for making peace here, rather argue that the seemingly insignificant issue of a single Cl ion in one of 100k structure models can lead to productive reflection about meaning and improvement of model description. Sorry for offending those in need for cozy comfort closing quotes. The answer is, as always, 42. HTC, BR (Happy To Confuse
Re: [ccp4bb] chloride or water
On Wed, Jan 21, 2015 at 10:17 AM, Keller, Jacob kell...@janelia.hhmi.org wrote: I see your point about not knowing that it’s a chloride, but I think you would agree that it is certainly more likely a chloride than map-noise, and perhaps more likely than water as well. Would you agree that chloride is the best guess, at least? No, I think I don't know is the most honest and scientifically robust answer. For those who insist on annotating every density blob, UNX atoms are the PDB's officially supported method for doing so (unless this has changed recently), or UNK/UNL for unknown amino acids and ligands. These are not without their own problems but they at least make both the presence of an atom and the uncertainty about its identity explicit. Since the PDB is certainly tainted by structures modeled in accordance with the “most likely” outlook, one now has to be cautious about all structures. This is true, but everyone else is just as sloppy is a poor excuse for further polluting the database. -Nat
Re: [ccp4bb] chloride or water
Hi Jacob, I would agree with Nat. I think with the 3A data and the local environment, putting a Cl there is only justified by the need of supressing a peak in the difference map. Yes the peak can really be a Cl or a water or a mixture of the two (I would like to do an experiment with my Cl containing data, which has Cl sites identified by iodide data, see what would happen when resolution is reduced and SNR is weakened). But we have to be aware that with 3A data there can be significant phase errors that lead to generation of features that are either not there or not so significant as it appears. In the past I have even seen a structure that puts a putative sugar (completely unknown type of glycosylation) in a blob in 3A structure because the authors thought the blob looked like the particular sugar residue. The practice of putting a ion or a small ligand simply for supressing difference peaks, to my view is not good practice for two reasons: 1) compared to protein residues, ions, and small ligands in low res cases, lack the geometric constraints and can be easily abused for the purpose of flattening the difference maps (admittedly a lot of water molecules is doing that, but we are told to be very careful with that already); 2) practically it makes searching/analysis of the structures in PDB a lot more difficult that it should be. It would be much easier for users to search an incomplete but reliable database than one that contains a mixture of real and wrong data, especially considering that not all users are experts on interpreting electron density maps or protein structures. Zhijie From: Keller, Jacob Sent: Wednesday, January 21, 2015 1:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water I reiterate that assigning a chloride is not “wild speculation” or “just making something up” in light of what we know about the situation. I see your point about not knowing that it’s a chloride, but I think you would agree that it is certainly more likely a chloride than map-noise, and perhaps more likely than water as well. Would you agree that chloride is the best guess, at least? What are the options for that blob, and what is the probability of each? I think you want to make sure people don’t get misled by it, which is a good point and a noble aspiration. I would argue that “not choosing” is here, as everywhere else, indeed choosing. And if you choose nothing here, you are almost certainly wrong, given the data. Some might be surprised or misled that a cavity like that would be totally empty, and the map density is unequivocal evidence that something is indeed there. So what now? Maybe a solution would be dummy atoms, maybe call them agnostons (agn) or something? Perhaps this is a basic disagreement about what a protein structure model represents, with one opinion being “that which we can rely upon confidently” and the other being “that which is most likely considering the data.” Each has advantages depending on one’s goals. Since the PDB is certainly tainted by structures modeled in accordance with the “most likely” outlook, one now has to be cautious about all structures. I prefer the latter since I would try to be responsible/skeptical enough to go back to the original crystal data before making important conclusions based on it. Maybe there should be a disclaimer printed in each PDB file… JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Wednesday, January 21, 2015 12:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.org wrote: Not sure why there is this level of suspicion about the poor halide when waters generally get assigned so haphazardly. I would say that there are probably more “wrong” waters in the PDB than wrong chlorides, but there’s not much fuss about that. Great, so leave it empty instead of just making something up. Perhaps future generations will figure out a more rigorous and quantitative method for handling such features than guessing based on screenshots posted to a mailing list. At this resolution water placement is difficult to justify anyway - and since neither the scattering properties nor the coordination distances are especially accurate, trying to assign chemical identity in the absence of any supporting information (for example anomalous data) is especially futile. (Although at least in this case the resolution is an obvious red flag - to a crystallographer, anyway - indicating that any lighter ions shouldn't be taken very seriously. Other biologists, of course, may be more trusting.) -Nat
Re: [ccp4bb] chloride or water
Quote: (I would like to do an experiment with my Cl containing data, which has Cl sites identified by iodide data, see what would happen when resolution is reduced and SNR is weakened) I just realized that this experiment has an inherent problem: artificially reducing the resolution cannot simulate the lack of order in low-diffracting crystals. Some large peaks in low-res structures in theory can arise simply because atoms in different ASU happened to have a high statistical appearance at that position, assuming that the phases are right. Correct me if I am wrong. Zhijie From: Zhijie Li Sent: Wednesday, January 21, 2015 2:12 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water Hi Jacob, I would agree with Nat. I think with the 3A data and the local environment, putting a Cl there is only justified by the need of supressing a peak in the difference map. Yes the peak can really be a Cl or a water or a mixture of the two (I would like to do an experiment with my Cl containing data, which has Cl sites identified by iodide data, see what would happen when resolution is reduced and SNR is weakened). But we have to be aware that with 3A data there can be significant phase errors that lead to generation of features that are either not there or not so significant as it appears. In the past I have even seen a structure that puts a putative sugar (completely unknown type of glycosylation) in a blob in 3A structure because the authors thought the blob looked like the particular sugar residue. The practice of putting a ion or a small ligand simply for supressing difference peaks, to my view is not good practice for two reasons: 1) compared to protein residues, ions, and small ligands in low res cases, lack the geometric constraints and can be easily abused for the purpose of flattening the difference maps (admittedly a lot of water molecules is doing that, but we are told to be very careful with that already); 2) practically it makes searching/analysis of the structures in PDB a lot more difficult that it should be. It would be much easier for users to search an incomplete but reliable database than one that contains a mixture of real and wrong data, especially considering that not all users are experts on interpreting electron density maps or protein structures. Zhijie From: Keller, Jacob Sent: Wednesday, January 21, 2015 1:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water I reiterate that assigning a chloride is not “wild speculation” or “just making something up” in light of what we know about the situation. I see your point about not knowing that it’s a chloride, but I think you would agree that it is certainly more likely a chloride than map-noise, and perhaps more likely than water as well. Would you agree that chloride is the best guess, at least? What are the options for that blob, and what is the probability of each? I think you want to make sure people don’t get misled by it, which is a good point and a noble aspiration. I would argue that “not choosing” is here, as everywhere else, indeed choosing. And if you choose nothing here, you are almost certainly wrong, given the data. Some might be surprised or misled that a cavity like that would be totally empty, and the map density is unequivocal evidence that something is indeed there. So what now? Maybe a solution would be dummy atoms, maybe call them agnostons (agn) or something? Perhaps this is a basic disagreement about what a protein structure model represents, with one opinion being “that which we can rely upon confidently” and the other being “that which is most likely considering the data.” Each has advantages depending on one’s goals. Since the PDB is certainly tainted by structures modeled in accordance with the “most likely” outlook, one now has to be cautious about all structures. I prefer the latter since I would try to be responsible/skeptical enough to go back to the original crystal data before making important conclusions based on it. Maybe there should be a disclaimer printed in each PDB file… JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Wednesday, January 21, 2015 12:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.org wrote: Not sure why there is this level of suspicion about the poor halide when waters generally get assigned so haphazardly. I would say that there are probably more “wrong” waters in the PDB than wrong chlorides, but there’s not much fuss about that. Great, so leave it empty instead of just making something up. Perhaps future generations will figure out a more rigorous and quantitative method for handling such features than guessing based on screenshots posted to a mailing list. At this resolution water placement is difficult to justify anyway - and since
Re: [ccp4bb] chloride or water
I reiterate that assigning a chloride is not “wild speculation” or “just making something up” in light of what we know about the situation. I see your point about not knowing that it’s a chloride, but I think you would agree that it is certainly more likely a chloride than map-noise, and perhaps more likely than water as well. Would you agree that chloride is the best guess, at least? What are the options for that blob, and what is the probability of each? I think you want to make sure people don’t get misled by it, which is a good point and a noble aspiration. I would argue that “not choosing” is here, as everywhere else, indeed choosing. And if you choose nothing here, you are almost certainly wrong, given the data. Some might be surprised or misled that a cavity like that would be totally empty, and the map density is unequivocal evidence that something is indeed there. So what now? Maybe a solution would be dummy atoms, maybe call them agnostons (agn) or something? Perhaps this is a basic disagreement about what a protein structure model represents, with one opinion being “that which we can rely upon confidently” and the other being “that which is most likely considering the data.” Each has advantages depending on one’s goals. Since the PDB is certainly tainted by structures modeled in accordance with the “most likely” outlook, one now has to be cautious about all structures. I prefer the latter since I would try to be responsible/skeptical enough to go back to the original crystal data before making important conclusions based on it. Maybe there should be a disclaimer printed in each PDB file… JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Wednesday, January 21, 2015 12:33 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: Not sure why there is this level of suspicion about the poor halide when waters generally get assigned so haphazardly. I would say that there are probably more “wrong” waters in the PDB than wrong chlorides, but there’s not much fuss about that. Great, so leave it empty instead of just making something up. Perhaps future generations will figure out a more rigorous and quantitative method for handling such features than guessing based on screenshots posted to a mailing list. At this resolution water placement is difficult to justify anyway - and since neither the scattering properties nor the coordination distances are especially accurate, trying to assign chemical identity in the absence of any supporting information (for example anomalous data) is especially futile. (Although at least in this case the resolution is an obvious red flag - to a crystallographer, anyway - indicating that any lighter ions shouldn't be taken very seriously. Other biologists, of course, may be more trusting.) -Nat
Re: [ccp4bb] chloride or water
No, I think I don't know is the most honest and scientifically robust answer. It will impress at least with its solemn humility, but in this case, you *do* know something, and that information makes the most likely answer that there is indeed something in that blob, and that something is probably chloride. This general scientific perspective should be understandable. It is more honest to model what you think is most likely rather than putting nothing, which has zero likelihood. Nothing is something also; hence, dishonesty. I know that you have written some software to do this type of thing automatically, and I guess you must have thought about the cautious vs. highest-likelihood perspectives quite a bit in so doing. I would guess then your software is very agnostic? Maybe you could try to see what the effect of different confidence thresholds (like requiring anomalous signal to identify halides(!)) would be on general model quality, say along the lines of pdb-redo. I predict that generally model quality will decrease with the high confidence cutoffs you recommend, and I hereby lay down the gauntlet for you to undertake this arduous and time-consuming project. For those who insist on annotating every density blob, UNX atoms are the PDB's officially supported method for doing so (unless this has changed recently), or UNK/UNL for unknown amino acids and ligands. These are not without their own problems but they at least make both the presence of an atom and the uncertainty about its identity explicit. Okay, so why don’t we recommend that this chloride be modeled as UNX? What’s the problem? (And why not make every water into UNX for good measure as well?) JPK
Re: [ccp4bb] chloride or water
On Wed, Jan 21, 2015 at 12:16 AM, Engin Özkan eoz...@uchicago.edu wrote: Carbon in chloride's coordination sphere? To me, it looks like you have serious vdW violations, and neither water nor chloride could go there. Halides can interact with carbon too - discussed in Dauter Dauter (2001) - although I think this is more common with iodide than chloride. But this instance is totally unconvincing without anomalous data. It would be better to leave it entirely empty than to put in something wildly speculative - there are far too many spurious chlorides in the PDB already, which of course makes it even more difficult to come up with general rules about binding patterns. -Nat
Re: [ccp4bb] chloride or water
The discrepancy between using the original mtz and an updated mtz is likely the result of the Fobs being placed on the Fcalc scale from Refmac output, not direct reading of Fcalc. -YW -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Wednesday, January 21, 2015 10:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water Check other Cl- in the pdb to see whether the bond lengths and VdW are an issue (wouldn't H2O have the same VdW issues?). I am thinking (this happened to me once by mistake) that maybe you are using the last updated mtz (output of refinement) to refine against rather than the original one. This for some reason can put weird blobs of density in cavities, and confused me once. I am still somewhat curious why this happens, since I would think Refmac would just use obs amplitudes (maybe it's reading Fcalc rather than Fobs?) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Carlos CONTRERAS-MARTEL Sent: Wednesday, January 21, 2015 9:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water Hi Rohit, What about your Rfree? what about your protein geometry? I will try to figure out this residual density at the realy latest step of the refinement. Because of your resolution, no anomalous signal available and Van der Waals problems it could be difficult for a referee believe in a Cl ion. That you probably wants to describe like close to you active site, so of some relevance for the activity. All the best Carlos -- Carlos CONTRERAS MARTEL, Ph.D. (CR1 CNRS) carlos.contreras-mar...@ibs.fr Bacterial Pathogenesis Group Institut de Biologie Structurale UMR5075 CEA-CNRS-Université Joseph Fourier Grenoble 1 IBS 71, avenue des Martyrs CS 10090 38044 Grenoble CEDEX 9 FRANCE tel : (+33) (0)4 57 42 86 41 http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/ http://www.ibs.fr/groups/bacterial-pathogenesis-group/
Re: [ccp4bb] chloride or water
I disagree with Nat—it is more likely that the blob is a chloride than a water, and dramatically more likely than a “nothing.” And it’s certainly not “wildly speculative,” considering that there is 200 mM Cl- in the crystallization cocktail, and the type of contacts it makes. Not sure why there is this level of suspicion about the poor halide when waters generally get assigned so haphazardly. I would say that there are probably more “wrong” waters in the PDB than wrong chlorides, but there’s not much fuss about that. I suspect that whatever reason you will give for not fussing about waters, you can apply that reason to chloride as well. I would concede that if some conclusion will be reached based on its being a chloride, one should be especially rigorous, (this would be true for water as well) but otherwise cannot see any reason for being so particular. If it fits the data best and is plausible, I say go for Cl-. Hopefully your reviewers will at least agree with each other! JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Wednesday, January 21, 2015 11:35 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water On Wed, Jan 21, 2015 at 12:16 AM, Engin Özkan eoz...@uchicago.edumailto:eoz...@uchicago.edu wrote: Carbon in chloride's coordination sphere? To me, it looks like you have serious vdW violations, and neither water nor chloride could go there. Halides can interact with carbon too - discussed in Dauter Dauter (2001) - although I think this is more common with iodide than chloride. But this instance is totally unconvincing without anomalous data. It would be better to leave it entirely empty than to put in something wildly speculative - there are far too many spurious chlorides in the PDB already, which of course makes it even more difficult to come up with general rules about binding patterns. -Nat
Re: [ccp4bb] chloride or water
On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.org wrote: Not sure why there is this level of suspicion about the poor halide when waters generally get assigned so haphazardly. I would say that there are probably more “wrong” waters in the PDB than wrong chlorides, but there’s not much fuss about that. Great, so leave it empty instead of just making something up. Perhaps future generations will figure out a more rigorous and quantitative method for handling such features than guessing based on screenshots posted to a mailing list. At this resolution water placement is difficult to justify anyway - and since neither the scattering properties nor the coordination distances are especially accurate, trying to assign chemical identity in the absence of any supporting information (for example anomalous data) is especially futile. (Although at least in this case the resolution is an obvious red flag - to a crystallographer, anyway - indicating that any lighter ions shouldn't be taken very seriously. Other biologists, of course, may be more trusting.) -Nat
Re: [ccp4bb] chloride or water
For those who insist on annotating every density blob, UNX atoms are the PDB's officially supported method for doing so (unless this has changed recently), or UNK/UNL for unknown amino acids and ligands. These are not without their own problems but they at least make both the presence of an atom and the uncertainty about its identity explicit. Okay, so why don’t we recommend that this chloride be modeled as UNX? What’s the problem? If you do that then what chemical type you are going to put into that rightmost column of ATOM record of PDB file? Note, that must be a valid chemical element symbol so that you can use such PDB file to calculate R-factors, maps and whatnot (Obviously, X would not work!). (And why not make every water into UNX for good measure as well?) Why not indeed, except that I would not call it water but Dummy Atom with an element type of your choice. Pavel
Re: [ccp4bb] chloride or water
If you do that then what chemical type you are going to put into that rightmost column of ATOM record of PDB file? Note, that must be a valid chemical element symbol so that you can use such PDB file to calculate R-factors, maps and whatnot (Obviously, X would not work!). Why, Cl- of course! Call it “Echols’ chloride.” (And why not make every water into UNX for good measure as well?) Why not indeed, except that I would not call it water but Dummy Atom with an element type of your choice. Because those blobs are most likely waters, it being 55 M or so! But admittedly, unless their being actually waters is used to assert something, I don’t think it matters that much. Most likely they are waters though. I think this will probably never happen, but maybe there could be a confidence value associated with each atom in structures a posteriori, although it might be difficult to find the right automatable criteria for this value. The element would be assigned by being the most likely one, and confidence assigned thereafter. Too much of a pain to implement, probably, and maybe not worth the trouble. Perhaps, though, Nat’s program could be used to do this. [ doi:10.1107/S1399004714001308http://dx.doi.org/10.1107/S1399004714001308 ]. JPK
Re: [ccp4bb] chloride or water
If you do that then what chemical type you are going to put into that rightmost column of ATOM record of PDB file? Note, that must be a valid chemical element symbol so that you can use such PDB file to calculate R-factors, maps and whatnot (Obviously, X would not work!). Why, Cl- of course! Call it “Echols’ chloride.” (And why not make every water into UNX for good measure as well?) Why not indeed, except that I would not call it water but Dummy Atom with an element type of your choice. Because those blobs are most likely waters, it being 55 M or so! But admittedly, unless their being actually waters is used to assert something, I don’t think it matters that much. Most likely they are waters though. Specifying chemical element type is important because it is used to pull out correct form-factors when the PDB file is used to calculate structure factors. Say a choice between O vs U may make a huge difference for R-factors! Pavel
Re: [ccp4bb] chloride or water
On Wed, Jan 21, 2015 at 12:24 PM, Keller, Jacob kell...@janelia.hhmi.org wrote: I think this will probably never happen, but maybe there could be a confidence value associated with each atom in structures a posteriori, although it might be difficult to find the right automatable criteria for this value. The element would be assigned by being the most likely one, and confidence assigned thereafter. Too much of a pain to implement, probably, and maybe not worth the trouble. Perhaps, though, Nat’s program could be used to do this. [ doi:10.1107/S1399004714001308 http://dx.doi.org/10.1107/S1399004714001308 ]. Unfortunately that implementation isn't really quantitative in the sense you describe - not for lack of interest on our part, but rather because coming up with a unified score based on many disparate and arguably incomplete criteria is quite difficult. The initial goal was to go after the low-hanging fruit of ions that are relatively obvious and/or whose identity is well-known, which would otherwise need to be placed manually. But as we tried to make clear in the paper, this isn't a substitute for common sense and prior knowledge. (By the way, for what it's worth, I think both Na and Cl are simultaneously over- and under-represented in the PDB - there are many spurious atoms, but at least as many that were overlooked and labeled as water. From the perspective of a methods developer, however, the false positives are much more of a pain to deal with in an automated workflow.) -Nat
Re: [ccp4bb] chloride or water
On 01/21/15 15:04, Pavel Afonine wrote: (And why not make every water into UNX for good measure as well?) Why not indeed, except that I would not call it water but Dummy Atom with an element type of your choice. there was a time long ago when budding crystallographers were taught to refer to solvent atoms rather than waters if there was doubt. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] chloride or water
Hi Rohit, What about your Rfree? what about your protein geometry? I will try to figure out this residual density at the realy latest step of the refinement. Because of your resolution, no anomalous signal available and Van der Waals problems it could be difficult for a referee believe in a Cl ion. That you probably wants to describe like close to you active site, so of some relevance for the activity. All the best Carlos -- Carlos CONTRERAS MARTEL, Ph.D. (CR1 CNRS) carlos.contreras-mar...@ibs.fr Bacterial Pathogenesis Group Institut de Biologie Structurale UMR5075 CEA-CNRS-Université Joseph Fourier Grenoble 1 IBS 71, avenue des Martyrs CS 10090 38044 Grenoble CEDEX 9 FRANCE tel : (+33) (0)4 57 42 86 41 http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/ http://www.ibs.fr/groups/bacterial-pathogenesis-group/
Re: [ccp4bb] chloride or water
Check other Cl- in the pdb to see whether the bond lengths and VdW are an issue (wouldn't H2O have the same VdW issues?). I am thinking (this happened to me once by mistake) that maybe you are using the last updated mtz (output of refinement) to refine against rather than the original one. This for some reason can put weird blobs of density in cavities, and confused me once. I am still somewhat curious why this happens, since I would think Refmac would just use obs amplitudes (maybe it's reading Fcalc rather than Fobs?) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Carlos CONTRERAS-MARTEL Sent: Wednesday, January 21, 2015 9:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water Hi Rohit, What about your Rfree? what about your protein geometry? I will try to figure out this residual density at the realy latest step of the refinement. Because of your resolution, no anomalous signal available and Van der Waals problems it could be difficult for a referee believe in a Cl ion. That you probably wants to describe like close to you active site, so of some relevance for the activity. All the best Carlos -- Carlos CONTRERAS MARTEL, Ph.D. (CR1 CNRS) carlos.contreras-mar...@ibs.fr Bacterial Pathogenesis Group Institut de Biologie Structurale UMR5075 CEA-CNRS-Université Joseph Fourier Grenoble 1 IBS 71, avenue des Martyrs CS 10090 38044 Grenoble CEDEX 9 FRANCE tel : (+33) (0)4 57 42 86 41 http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/ http://www.ibs.fr/groups/bacterial-pathogenesis-group/
Re: [ccp4bb] chloride or water
Hi Rohit, you might be interested in the following paper: Buried chloride stereochemistry in the Protein Data Bank BMC Structural Biology 2014, 14:19 doi:10.1186/s12900-014-0019-8 http://www.biomedcentral.com/1472-6807/14/19 Best, Oliviero On Tue, January 20, 2015 10:53, Eleanor Dodson wrote: Also it just might be useful to do an anomalous Fourier - Cl has a weak signal, but may be visible if the phases are good. cf any peaks to ones over the S atoms to get a measure of what you might expect- Eleanor On 20 January 2015 at 07:17, Robbie Joosten r.joos...@nki.nl wrote: Hi Rohit, The pictures you sent are not that informative. There are a few things you can use to distinguish chloride from water. Chloride prefers nitrogens over oxygens as ligands. Also the coordination lengths are higher than those of water, typically between 3 and 3.5Å. Usually, the coordination is not as pretty as that of cations. HTH, Robbie -- Van: rohit kumar rohit...@gmail.com Verzonden: 20-1-2015 08:05 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: [ccp4bb] chloride or water Dear all, I am solving a data of 3.0 Å resolution. In the active site we found an unidentified blob. The crystallization condition is (PEG 3350-25%, Naformate-200mM, MES (100mM)-6.5pH, and Nacl-200mM). Can anyone suggest what it could be? If I suppose that, it is chloride, how could someone differentiate between a chloride moiety and water. below I am showing the coot figure in two different orientations. Thanks in advance... [image: Inline image 1] [image: Inline image 2] -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
Re: [ccp4bb] chloride or water
Try each one, refine, compare resulting b-factors and bond lengths to surrounding atoms. It’s a chloride though. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhijie Li Sent: Tuesday, January 20, 2015 2:53 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] chloride or water Hi Rohit, If it is of some significance you may consider replacing NaCl with KI or NaI in your crystallization condition and collect a dataset at 1.5-2 Angstrom. The Iodide anomalous signal might be of some help for identifying Chloride binding sites. Without further evidence for a Cl ion, unless the environment strongly suggests an anion binding site (as what Robbie said) my personal view is that it is more proper to put a water there. Zhijie From: rohit kumarmailto:rohit...@gmail.com Sent: Tuesday, January 20, 2015 1:59 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] chloride or water Dear all, I am solving a data of 3.0 Å resolution. In the active site we found an unidentified blob. The crystallization condition is (PEG 3350-25%, Naformate-200mM, MES (100mM)-6.5pH, and Nacl-200mM). Can anyone suggest what it could be? If I suppose that, it is chloride, how could someone differentiate between a chloride moiety and water. below I am showing the coot figure in two different orientations. Thanks in advance... -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
Re: [ccp4bb] chloride or water
Hi Rohit, If it is of some significance you may consider replacing NaCl with KI or NaI in your crystallization condition and collect a dataset at 1.5-2 Angstrom. The Iodide anomalous signal might be of some help for identifying Chloride binding sites. Without further evidence for a Cl ion, unless the environment strongly suggests an anion binding site (as what Robbie said) my personal view is that it is more proper to put a water there. Zhijie From: rohit kumar Sent: Tuesday, January 20, 2015 1:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] chloride or water Dear all, I am solving a data of 3.0 Å resolution. In the active site we found an unidentified blob. The crystallization condition is (PEG 3350-25%, Naformate-200mM, MES (100mM)-6.5pH, and Nacl-200mM). Can anyone suggest what it could be? If I suppose that, it is chloride, how could someone differentiate between a chloride moiety and water. below I am showing the coot figure in two different orientations. Thanks in advance... -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
