Re: [ccp4bb] Jrh input Re: [ccp4bb] what to do with disordered side chains
Dear Ed, Many thanks for this careful explanation, which I appreciate. I realise in my own practise on such matters I have two situations :- (i) where, albeit limited, electron density evidence, coupled with chemical evidence, leads to an attempted model atomic fit but the B factors there sky rocket. (ii) there is no electron density evidence and even though the chemical evidence is sound one can in fact add nothing. Here I simply concede that there is nothing one can do. The issue here is not whether to keep these atoms but simply you really cannot make a start finding them. Greetings, John On Fri, Apr 1, 2011 at 4:03 PM, Ed Pozharski epozh...@umaryland.edu wrote: Dear John, there may be reasons to disagree with both options. This has been a recurring discussion for many years, and in my mind the most convincing arguments for both sides are as follows: Keepers: I know the side chain is there and the high ADP is a good approximation of reality. Removing atoms causes such a mess for the end user. Deleters: We don't model missing loops, termini, ligands and waters when there is no density, and side chains should not be treated differently. Most end users think ADP is a nucleotide and will over-interpret the model. I am a keeper when it comes to end user treatment, but a recently converted deleter when it comes to modeling (a rather stressful position). So I am not taking sides really, but rather looking for a middle way. (Have to admit that my secret goal was to knock down the zero occupancy fallacy :) Perhaps these ideas are worth exploring: 1. Provide dual representation - a crystallographic model and an end-user model, both downloadable from the PDB. 2. Model missing side chains NMR-way 3. A new data file format is needed (mmCIF?) that combines atomic model with electron density, and visualization/analysis software shall be modified to always utilize the experimental data 4. Implement reduced ADP restraints for disordered side chains to further reduce model bias But ultimately, as long as experimental data is deposited, I believe that people are free to interpret their data the way they see fit. Others are then free to look at the electron density and become outraged at the interpretation. Cheers, Ed. On Thu, 2011-03-31 at 23:25 +0100, Jrh wrote: Dear Ed, Thankyou for this and apologies for late reply. If one has chemical evidence for the presence of residues but these residues are disordered I find the delete atoms option disagreeable. Such a static disorder situation should be described by a high atomic displacement parameter, in my view. (nb the use of ADP is better than B factor terminology). Yours sincerely, John Prof John R Helliwell DSc -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs -- Professor John R Helliwell DSc
[ccp4bb] Call for paper
Dear colleagues, The Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia is organising the International Interdisciplinary Science Conference 2011, on “Bioinformatics: An Interface between Computer Science and Biology. The Conference will be held during November 15-17, 2011 in the University premises. The purpose of this conference is to provide an opportunity to Ph. D. scholars and M. Sc. students of both basic sciences and life sciences to listen to and interact with established researcher s (Bioinformatists, System Biologists, Microbiologists, Immunologists, Molecular Biologists and Clinicians) who are making use of computer science in understanding biological problems. In addition to this, they will also have an opportunity to meet representatives and policy makers from the pharmaceutical and biotechnological industries. Your research scholars and M. Sc. students are encouraged to send their abstracts for poster presentation. Some of the accepted posters will be selected for oral presentation. For details about registration and abstract submission in the I-ISC2011, please visit our website: http://www.i-isc.com/ With warm regards, Yours sincerely, Dr. M. I. Hassan Organizing Secretary I-ISC-2011 E-mail: mihas...@jmi.ac.in =Md. Imtiyaz Hassan, Ph.D. Assistant Professor (Biophysics) Centre for Interdisciplinary Research In Basic Saciences Jamia Millia Islamia New Delhi 10025, INDIA TeleFax: +91 11 26983409 Mob-9311323414 9990323217 E-mail: mihas...@jmi.ac.in http://www.jmi.nic.in/cirbs/cirbs.htm
Re: [ccp4bb] what to do with disordered side chains
If these users exist (I don't doubt that they do), then they would also might think that lysine residues sometimes look identical to alanine - if the atoms after beta carbon of the lysine are deleted in the PDB due to lack of density. So, I guess, if one's objectives in solving structures are to provide these users with coordinates that they could us, then it would make more sense to me to find out what one's customers want, rather than speculating about it. Or at least, train one's customers how to use one's products - I believe that's what people in business do. However, I think that many people solve structures for their own consumption - they are their own customers - therefore, it's really up to them to cook it anyway they find most tasteful. Others can agree or disagree, but we know that not everybody has the same taste. Cheers, Quyen On Apr 3, 2011, at 12:54 AM, Prof. Joel L. Sussman wrote: I think Frances is right, i.e. most non crystallographers ignore anything beyond the x, y, z coordinates (i.e. beyond column 54) [as Frances wrote]. Thus if a crystallographer put in coords that he/she does NOT see, even with OCC=0, or an enormously large Bfactor, these coords are usually treated in just the same way that experimentally observed coords are treated. Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software that does its voodoo on ATOM/HETATM records and pays absolutely no attention to anything beyond the x, y, z coordinates (i.e. beyond column 54). Frances Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Sat, 2 Apr 2011, Jacob Keller wrote: I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Some users might pull up the structure, see those atoms, and think their positions were based on data, which they were not, and then draw conclusions based on them. I agree that occ=0 is tantamount to the suggestion you queried, however. A somewhat key question might be: across the various molecular visualization programs, what is the default way to handle atoms with occ=0? Perhaps those programs might be the best place to fix the problem... JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman Concur. BMC p 680 'How to handle missing parts' Best wishes, BR On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software that does its voodoo on ATOM/HETATM records and pays absolutely no attention to anything beyond the x, y, z coordinates (i.e. beyond column 54). Frances Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Sat, 2 Apr 2011, Jacob Keller wrote: I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Some users might pull up the structure, see those atoms, and think their positions were based on data, which they were not, and then draw conclusions based on them. I agree that occ=0 is tantamount to the suggestion you queried, however. A somewhat key question might be: across the various molecular visualization programs, what is the default way to handle atoms with occ=0? Perhaps those programs might be the best place to fix the problem... JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
On Sunday, April 03, 2011, Jacob Keller wrote: To the delete-the-atom-nik's: do you propose deleting the whole residue or just the side chain? Omit the atoms beyond CB for which there is no apparent density. Always place CB if the backbone trace is reasonable, because its location is fixed a priori by known stereochemistry. As a practical matter, I use Coot's stub command. Ethan I can understand deleting the whole residue, but deleting only the side chain seems to me to be placing a stumbling block also, and even possibly confusing for an experienced crystallographer: the .pdb says lys but it looks like an ala? Which is it? I could imagine a lot of frustration-hours arising from this practice, with people cross-checking sequences, looking in the methods sections for mutations... JPK On Sun, Apr 3, 2011 at 11:42 AM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman Concur. BMC p 680 ‘How to handle missing parts’ Best wishes, BR On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software that does its voodoo on ATOM/HETATM records and pays absolutely no attention to anything beyond the x, y, z coordinates (i.e. beyond column 54). Frances Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Sat, 2 Apr 2011, Jacob Keller wrote: I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Some users might pull up the structure, see those atoms, and think their positions were based on data, which they were not, and then draw conclusions based on them. I agree that occ=0 is tantamount to the suggestion you queried, however. A somewhat key question might be: across the various molecular visualization programs, what is the default way to handle atoms with occ=0? Perhaps those programs might be the best place to fix the problem... JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
Hi, it is quite possible to truncate say Lys residue isnt it? so why not do this, this doesn't change the identity of the residue but precisely draws attention to the fact that atoms are missing due to lack of density. And if you click on an atom in Pymol, at least i dont see the b-factor displayed anywhere - i would suspect its the same case with other mol. graphics visualization software to fair extent. or its some small print somewhere... + can you actually tell by just looking at the B-factor whether there is any density or not? if the wilson b is high i suspect you can see density and the B-factor will be high where as if Wilson B is low same b-factor will probably mean you dont see density at same sigma cutoff/contour level. or i may be wrong but suspect this is the casewhich is why i think its better probably to truncate (not to ala/gly, but to truncate) them if you don't see the density for the side chain at all. OR model the 5 most like conformers then - or 4 - 6 ? 3? well, this can go on forever --or rather hopefully NOT, but really i don't think this quite so simple as what comes to B-factors and later analysis ---in particular if that will be in anyway automated and will deal with say a larger set of coordinate files. is it really a good idea to leave an active site residue side chain with high B (=no density what so ever) in, in _one_ defined conformation? i am not so dead certain... cheers, Tommi On Apr 3, 2011, at 9:01 PM, Boaz Shaanan wrote: The original posting that started this thread referred to side- chains, as the subject still suggests. Do you propose to omit only side-chain atoms, in which case you end up with different residues, as pointed out by quite a few people,or do you suggest also to omit the main-chain atoms of the problematic residues ? Besides, as mentioned by Phoebe and others, many users (non- crystallographers) of PDB's know already the meaning of the B- factor and will know how to interpret a very high B. It is our task (the crystallographers) to enllighten those who don't know what the B column in a PDB entry stands for. I certainly do and I'm sure many of us do so too. I voted for high B and would vote for it again, if asked. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Sunday, April 03, 2011 7:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman Concur. BMC p 680 ‘How to handle missing parts’ Best wishes, BR On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software that does its voodoo on ATOM/HETATM records and pays absolutely no attention to anything beyond the x, y, z coordinates (i.e. beyond column 54). Frances Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.commailto:f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Sat, 2 Apr 2011, Jacob Keller wrote: I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Some users might pull up the structure, see those atoms, and think their positions were based on data, which they were not, and then draw conclusions based on them. I agree that occ=0 is tantamount to the suggestion
Re: [ccp4bb] what to do with disordered side chains
Clearly there are strong feelings held by the advocates of the several solutions to the problem of what to do about atoms that cannot be reliably placed based on the electron density map. I certainly understand since I passionately support my own favorite solution. Why is it that a community of generally reasonable people keep coming back to this same issue and yet fail to find a solution that can reach some kind of consensus? My 2 cents on this, more fundamental, issue: A model created by someone who believes that all atoms (for a residue with any atoms) must be built will contain two kinds of atoms. Those placed based on the appearance of the electron density and those placed in some convenient location simply to fill out the atom count. I think most everyone agrees that a full residue is a convenience for some users of our models. What those of us who favor partial models want is an absolutely clear distinction between the two classes of atoms. All this needs is a bit. Literally, one bit of data that flags those atoms added to the model simply to complete the set. Why can't we come to a solution that satisfies? Because we continue to use a non-extensible file format that does not allow us a place to put this bit. Some people want to put the bit in the occupancy column by defining a special value (occ=0) that would be the flag. Some people want to put it in the B factor column by defining a special value there (a couple possibilities here, B=1000.00, B=500.00, B varying but larger than that of any atom built into density). The B factor and occupancy columns in the PDB file have been precisely defined back when the mmCIF dictionary was created and to change their definitions now would require opening that process again. I am pretty sure that committee in charge will never allow a definition for these items that includes the phrase ... except when the value is equal too You can't run a database that way. Each piece of information has to have its own tag and definition. Once it is defined we can embrace the task of educating software developers and our collaborators who use our models in its meaning. There is just no place to put this bit in a PDB format file. mmCIF - its trivial. PDB format - no way. As long as we insist that this format is the preferred means of distributing our models we will continue to return to this argument again and again with no possibility of coming to a solution. Dale Tronrud P.S. I've even thought about using the model of the REMARK statement, where all sorts of information have been added by the hack of standardized remarks. I thought that one could create a standardized footnote that would mark the atoms as imaginary. I found that, unfortunately, footnotes were removed from the PDB format many years ago. On 4/3/2011 11:01 AM, Boaz Shaanan wrote: The original posting that started this thread referred to side-chains, as the subject still suggests. Do you propose to omit only side-chain atoms, in which case you end up with different residues, as pointed out by quite a few people,or do you suggest also to omit the main-chain atoms of the problematic residues ? Besides, as mentioned by Phoebe and others, many users (non-crystallographers) of PDB's know already the meaning of the B-factor and will know how to interpret a very high B. It is our task (the crystallographers) to enllighten those who don't know what the B column in a PDB entry stands for. I certainly do and I'm sure many of us do so too. I voted for high B and would vote for it again, if asked. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Sunday, April 03, 2011 7:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman Concur. BMC p 680 ‘How to handle missing parts’ Best wishes, BR On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software
Re: [ccp4bb] OT: PCR instrument
if the PCR machine is just to be used for standard sub-cloning (amplifying fragments from other plasmids, cloned cDNA etc.), I would go for the cheapest one I could find. I guess there are few crystallography projects were the first PCR step turned out to be the most difficult. For more sophisticated applications there are probably forums where more knowledgeable people reside (on PCR that is...) Mark Quoting Bernhard Rupp (Hofkristallrat a.D.): Dear All, I was polled for a recommendation for a good PCR instrument, but I am not much of a molecular biology person - if someone could please help and kindly send some recommendations to Eric W. Reinheimer ewreinhei...@csupomona.edu Best regards, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - No animals were hurt or killed during the production of this email. - Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] what to do with disordered side chains
I vote for the electron density irrespective of side chains, main chains, ligands, dark matter. The PDB is a collection of experimentally determined structures per its own definition. If density supports it high B is fine - B-factor simply is a parameter of a probability distribution. If you extend that to no density - it becomes problematic. After all, coordinates imply that an atom is actually at some specified place with a certain probability. We may know that the atom necessarily has to be someplace lest it got chewed off for some reason. The experiment just tells you that you do not know where the atom is. Or in Rumsfeldic: Better a known unknown than a unknown known. Cheers, BR -Original Message- From: Boaz Shaanan [mailto:bshaa...@exchange.bgu.ac.il] Sent: Sunday, April 03, 2011 11:02 AM To: hofkristall...@gmail.com; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] what to do with disordered side chains The original posting that started this thread referred to side-chains, as the subject still suggests. Do you propose to omit only side-chain atoms, in which case you end up with different residues, as pointed out by quite a few people,or do you suggest also to omit the main-chain atoms of the problematic residues ? Besides, as mentioned by Phoebe and others, many users (non-crystallographers) of PDB's know already the meaning of the B-factor and will know how to interpret a very high B. It is our task (the crystallographers) to enllighten those who don't know what the B column in a PDB entry stands for. I certainly do and I'm sure many of us do so too. I voted for high B and would vote for it again, if asked. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Sunday, April 03, 2011 7:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains Thus my feeling is that if one does NOT see the coords in the electron density, they should NOT be included, and let someone else try to model them in, but they should be aware that they are modeling them. Joel L. Sussman Concur. BMC p 680 'How to handle missing parts' Best wishes, BR On 3 Apr 2011, at 06:15, Frances C. Bernstein wrote: Doing something sensible in the major software packages, both for graphics and for other analysis of the structure, could solve the problem for most users. But nobody knows what other software is out there being used by individuals or small groups. And the more remote the authors of that software are from protein structure solution the more likely it is that they have not/will not properly handle atoms with zero occupancy or high B values, for example. I am absolutely positive that there is software that does its voodoo on ATOM/HETATM records and pays absolutely no attention to anything beyond the x, y, z coordinates (i.e. beyond column 54). Frances Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.commailto:f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Sat, 2 Apr 2011, Jacob Keller wrote: I guess I missed it in the flurry of replies to this thread over the last few days, but what exactly is so terrible about keeping the atoms (since you have chemical evidence from protein sequence that they are there, and even if there is X-ray damage they were originally there and are likely still there in a subset of the molecules), but changing occupancy to zero as an acknowledgment that your data does not provide evidence to support a specific atomic position for these atoms? Some users might pull up the structure, see those atoms, and think their positions were based on data, which they were not, and then draw conclusions based on them. I agree that occ=0 is tantamount to the suggestion you queried, however. A somewhat key question might be: across the various molecular visualization programs, what is the default way to handle atoms with occ=0? Perhaps those programs might be the best place to fix the problem... JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
Most non-structural users are familiar with the sequence of the proteins they are studying, and most software does at least display residue identity if you select an atom in a residue, so usually it is not necessary to do any cross checking besides selecting an atom in the residue and seeing what its residue name is. The chance of somebody misinterpreting a truncated Lys as Ala is, in my experience, much much lower than the chance they will trust the xyz coordinates of atoms with zero occupancy or high B factors. What worries me the most is somebody designing a whole biological experiment around an over-interpretation of details that are implied by xyz coordinates of atoms, even if those atoms were not resolved in the maps. When this sort of error occurs it is a level of pain and wasted effort that makes the pain associated with having to build back in missing side chains look completely trivial. As long as the PDB file format is the way users get structural data, there is really no good way to communicate atom exists with no reliable coordinates to the user, given the diversity of software packages out there for reading PDB files and the historical lack of any standard way of dealing with this issue. Even if the file format is hacked there is no way to force all the existing software out there to understand the hack. A file format that isn't designed with this sort of feature from day one is not going to be fixable as a practical matter after so much legacy code has accumulated. -Eric On Apr 3, 2011, at 2:20 PM, Jacob Keller wrote: To the delete-the-atom-nik's: do you propose deleting the whole residue or just the side chain? I can understand deleting the whole residue, but deleting only the side chain seems to me to be placing a stumbling block also, and even possibly confusing for an experienced crystallographer: the .pdb says lys but it looks like an ala? Which is it? I could imagine a lot of frustration-hours arising from this practice, with people cross-checking sequences, looking in the methods sections for mutations... JPK
Re: [ccp4bb] what to do with disordered side chains
Well, what about getting the default settings on the major molecular viewers to hide atoms with either occ=0 or bcutoff (novice mode?)? While the b cutoff is still be tricky, I assume we could eventually come to consensus on some reasonable cutoff (2 sigma from the mean?), and then this approach would allow each free-spirited crystallographer to keep his own preferred method of dealing with these troublesome sidechains and nary a novice would be led astray JPK On Sun, Apr 3, 2011 at 2:58 PM, Eric Bennett er...@pobox.com wrote: Most non-structural users are familiar with the sequence of the proteins they are studying, and most software does at least display residue identity if you select an atom in a residue, so usually it is not necessary to do any cross checking besides selecting an atom in the residue and seeing what its residue name is. The chance of somebody misinterpreting a truncated Lys as Ala is, in my experience, much much lower than the chance they will trust the xyz coordinates of atoms with zero occupancy or high B factors. What worries me the most is somebody designing a whole biological experiment around an over-interpretation of details that are implied by xyz coordinates of atoms, even if those atoms were not resolved in the maps. When this sort of error occurs it is a level of pain and wasted effort that makes the pain associated with having to build back in missing side chains look completely trivial. As long as the PDB file format is the way users get structural data, there is really no good way to communicate atom exists with no reliable coordinates to the user, given the diversity of software packages out there for reading PDB files and the historical lack of any standard way of dealing with this issue. Even if the file format is hacked there is no way to force all the existing software out there to understand the hack. A file format that isn't designed with this sort of feature from day one is not going to be fixable as a practical matter after so much legacy code has accumulated. -Eric On Apr 3, 2011, at 2:20 PM, Jacob Keller wrote: To the delete-the-atom-nik's: do you propose deleting the whole residue or just the side chain? I can understand deleting the whole residue, but deleting only the side chain seems to me to be placing a stumbling block also, and even possibly confusing for an experienced crystallographer: the .pdb says lys but it looks like an ala? Which is it? I could imagine a lot of frustration-hours arising from this practice, with people cross-checking sequences, looking in the methods sections for mutations... JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
I guess, most hydrophilic side chains on the surface are flexible, they don't keep the same conformation. If you cut those side chains off, the surface will be looking pretty hydrophobic and misleading (and very horrible). I prefer to see them intact. I know, most of them are flexible and don't have one exact position, but it's OK. I know they are there not far from the main chain. Usually, their exact position is irrelevant. Maia Jacob Keller wrote: Well, what about getting the default settings on the major molecular viewers to hide atoms with either occ=0 or bcutoff (novice mode?)? While the b cutoff is still be tricky, I assume we could eventually come to consensus on some reasonable cutoff (2 sigma from the mean?), and then this approach would allow each free-spirited crystallographer to keep his own preferred method of dealing with these troublesome sidechains and nary a novice would be led astray JPK On Sun, Apr 3, 2011 at 2:58 PM, Eric Bennett er...@pobox.com wrote: Most non-structural users are familiar with the sequence of the proteins they are studying, and most software does at least display residue identity if you select an atom in a residue, so usually it is not necessary to do any cross checking besides selecting an atom in the residue and seeing what its residue name is. The chance of somebody misinterpreting a truncated Lys as Ala is, in my experience, much much lower than the chance they will trust the xyz coordinates of atoms with zero occupancy or high B factors. What worries me the most is somebody designing a whole biological experiment around an over-interpretation of details that are implied by xyz coordinates of atoms, even if those atoms were not resolved in the maps. When this sort of error occurs it is a level of pain and wasted effort that makes the pain associated with having to build back in missing side chains look completely trivial. As long as the PDB file format is the way users get structural data, there is really no good way to communicate atom exists with no reliable coordinates to the user, given the diversity of software packages out there for reading PDB files and the historical lack of any standard way of dealing with this issue. Even if the file format is hacked there is no way to force all the existing software out there to understand the hack. A file format that isn't designed with this sort of feature from day one is not going to be fixable as a practical matter after so much legacy code has accumulated. -Eric On Apr 3, 2011, at 2:20 PM, Jacob Keller wrote: To the delete-the-atom-nik's: do you propose deleting the whole residue or just the side chain? I can understand deleting the whole residue, but deleting only the side chain seems to me to be placing a stumbling block also, and even possibly confusing for an experienced crystallographer: the .pdb says lys but it looks like an ala? Which is it? I could imagine a lot of frustration-hours arising from this practice, with people cross-checking sequences, looking in the methods sections for mutations... JPK
Re: [ccp4bb] OT: PCR instrument
I am too poor to afford cheap things (I am not sure where quote comes from but it's relevant). The answer depends on whether time is considered expensive or not. Sometimes (e.g. when free labor such as graduate students) time is not an issue. And sometimes it is. Reagent costs are fairly low now. For time, one cannot beat the Robocycler. It's a funny looking design, but it's really fast and quite accurate (since the blocks do not change temperature throughout the process). Artem On Sun, Apr 3, 2011 at 2:42 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: if the PCR machine is just to be used for standard sub-cloning (amplifying fragments from other plasmids, cloned cDNA etc.), I would go for the cheapest one I could find. I guess there are few crystallography projects were the first PCR step turned out to be the most difficult. For more sophisticated applications there are probably forums where more knowledgeable people reside (on PCR that is...) Mark Quoting Bernhard Rupp (Hofkristallrat a.D.): Dear All, I was polled for a recommendation for a good PCR instrument, but I am not much of a molecular biology person - if someone could please help and kindly send some recommendations to Eric W. Reinheimer ewreinhei...@csupomona.edu Best regards, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - No animals were hurt or killed during the production of this email. - Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
