Re: [ccp4bb] Citations in supplementary material
I don't wish to vear away from Victor's thrust with starting this thread and I would happily sign the petition you suggest. But I feel I should respond to the assertions about 'problems of peer review' at least with respect to Journals of my experience. Some 'Editor handling of submissions' statistics should help quantify such matters. These are a matter of public record re my IUCr Journals submission handling statistics ie therefore not confidential and which basically are:- approx 1000 article submissions; my rejection rate 20%; appeals against my rejections 0.5%; As Editor in Chief of Acta Cryst between 1996 to 2005 I received three appeals (out of approx tens of thousands of submissions through all Coeditors); I rejected these three. [My judgements were confidential re the details.] I can add that for the 2000 referees' reports or so for my article handling of submissions, that colleagues have kindly supplied to my Editor requests, problems involve:- about 1% where the report is 'publish as is' AND without any commendation given; these are in effect not terribly useful reports to me as an Editor. Another problem, which is growing, is the number of declines to my invites to referee (around 10%). Even worse are the no replies at all from invited referees as time is lost to the authors who rightly expect as prompt as possible handling. Re your points I offer replies as follows:- Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement takes a temporary leave of abesnse. JRH reply:- Such reports would be easy to spot and are not a problem in my experience and so resort to double blind review is not necessary in my experience. 2. 'preferred reviewers'. a double edged sword .. think about it. JRH reply; these are not so commonly offered suggestions by authors in fact and where they are one can follow or decide against (see point 1). 3. too much power of decision on editors (professional or academic) being able to reject papers without peer-review in many journals. JRH reply;This approach, 'insufficent general interest' is for the magazines we know and yet still love. 4. Bad refereeing - sometimes I wonder if people read the paper. JRH reply;Such reports are very few and obvious. The other categories above are more common (ie 'publish as is' category). 5. Lack of referee expertise: you get papers these days with: a structure, some biochemistry, some SAXS, some biophysics, and a cell based assay. Two or three people being able to pick up all the mistakes is very unlikely. JRH reply; Papers can be challenging re content and your example here is a good one. Other chalenging cases are where they include a lot of maths. That said peer review does its best but can occasionally fail; this level of failure can be measured by the number of criticism articles or formal retractions. These are also very few, but it is true, not zero. Yours sincerely, John On Thu, Nov 18, 2010 at 10:47 AM, Anastassis Perrakis a.perra...@nki.nl wrote: On Nov 18, 2010, at 11:18, James Stroud wrote: The future of publishing will be (1) Publish your own work (2) Peer review by the entire community Although I have been remarkably bad at predicting the future, I still like attempting to do so ...! This will not happen ...! ;-) To be honest, I am not even sure its a great idea ... Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement takes a temporary leave of abesnse. 2. 'preferred reviewers'. a double edged sword .. think about it. 3. too much power of decision on editors (professional or academic) being able to reject papers without peer-review in many journals. 4. Bad refereeing - sometimes I wonder if people read the paper. 5. Lack of referee expertise: you get papers these days with: a structure, some biochemistry, some SAXS, some biophysics, and a cell based assay. Two or three people being able to pick up all the mistakes is very unlikely. Having outlines these, I can see ways that all can be amplified if you just publish all your work, and anybody can comment on it: Pairing to the above problems, you just amplify them: 1. Even more tempting to earn brownie points online! 2. you can ask your friends or I can ask your enemies to review 3. the other way around: far too many things out... how to filter ? 4. Lack of 'obligation', or even fear to make yourself look like a fool to the editors, will make commenting even more sloppy 5. People that think they are experts dwell on meaningless technicalities. Peer review is like democracy, its the worst publication system we can have, except the ones that have been tried or suggested ... A. (3) Citation = Link #3 makes it work. Give it 25 years. The journals won't be in the position to lobby
Re: [ccp4bb] origin_shift in polar space group
Have you tried csymmatch -pdbin-ref one.pdb -pdbin two.pdb That will move chains to match asfar as possible, using sym ops and allowedorigin shifts to generate the best fit. Eleanor On 11/18/2010 12:26 PM, Ian Tickle wrote: OK now I understand. I couldn't find the script 'origin.com' you mentioned in the examples directory (at least from the filename I assume it's a script, not a MS-DOS program!), but it doesn't matter, I see the problem now. AFAIK there isn't a script in CCP4 that will do what you want entirely automatically, because it's actually quite a complicated problem in the completely general case of N molecules per a.u., though undoubtedly it could easily be scripted for the relatively simple case of 2 mols per a.u.. I'm assuming you don't simply want to superpose the molecules just for structural comparison purposes, you want to superpose the entire *crystals*, so that the calculated structure factors and hence the R factors (values) remained unchanged for the transformed structure. This means you can't use just arbitrary rotation/translation operators as would be generated by superposition programs such as SSM, you have to restrict it to crystallographically-allowed origin shifts. There are various programs which will do this, I wrote one called 'reforigin' but there are others which will do the same thing, and which have been mentioned in previous postings. So what you have to do is superpose the two 'A' molecules using reforigin or whatever (remember, as long as it applies only crystallographically-allowed origin shifts). There is of course a problem here: the chain ID 'A' is only an arbitrary label, so there's a 50% chance that the molecule you called 'A' in structure 1 might be called 'B' in structure 2 (and vice versa). This means you have to try both possibilities! Now you see why it gets complicated in the general case with molecules 'A', 'B', 'C', 'D' ... you have to try all combinations! While you are superposing A/2 on A/1 (or B/2 on A/1) you must also transform the other chain B/2 (or A/2) using the *same* operator (I think the program does this for you, or at least it will print the matrix that was used for the 1st pair) - you must not superpose it independently. Finally you need to transform the other molecule B/2 (or A/2) in the example above. For this you can only use space-group symmetry operators - you get only one chance to use the allowed origin shifts with the first pair of molecules, after that the origin is completely determined for the entire structure, hence only space-group symmetry can be used to transform subsequent pairs. For this I find it easiest just to view the structure on the graphics, work out which is appropriate space-group operator and apply it just to the 2nd molecule using PDBSET. Hope this is all clear - there are many traps here for the unwary! Cheers -- Ian On Thu, Nov 18, 2010 at 10:55 AM, Rojan Shrestharo...@riken.jp wrote: Hello Ian: I am afraid that whether my problem is not clear to you. Here is brief description of the problem. When I tried to superimpose two structures having two or more copies in ASU for polar space group using symmetric operator, for one copies it used one origin and for next, another origin is used. So there is origins shift problem. Here is an example: applying 0.50 0.50 -0.69 Y,-X,3/4+Z to chain A applying 1.50 0.50 0.61 X,Y,Z to chain B WARNING: ./input.pdb chain B is on a different origin! I used origin.com to superimpose two models. Now I hope you get the insight of my problem. Do you have any idea to solve this problem? Regards, Rojan -Original Message- From: Ian Tickle [mailto:ianj...@gmail.com] Sent: Thursday, November 18, 2010 7:42 PM To: ro...@riken.jp Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] origin_shift in polar space group HI Rojan, I'm not entirely clear that there is a problem. After superposition any origin shift that may have been present is removed: doesn't that solve your problem? Cheers -- Ian On Thu, Nov 18, 2010 at 10:11 AM, Rojan Shrestharo...@riken.jp wrote: Hello: In polar space group when the two or more copies molecules are superimposed, the origin is shifted. Does anybody have the solution to tackle this problem? Regards, Rojan
Re: [ccp4bb] Citations in supplementary material
Dear John, I did not have the IUCr journals specifically in mind while making these remarks. Quite the contrary, I think due to you and other colleagues and friends, they are run competently and to the benefit of the community and of science at large. If I may though offer an opinion about the peer review system in IUCr journals, I personally find the concept that the authors know the identity of the managing editor, wrong. I am sure that in the majority of cases its not a problem, but often a managing editor can hesitate to communicate a negative referee report to e.g. an old colleague or good friend, whose manuscript she/he is handling, even when one of the referees is negative. I much prefer the system of e.g. Proteins (where I act as a managing editor), where authors never learn the identity of the managing editor far more comfortable (there is a few people that I would rather prefer if they don't know for sure that I rejected their paper), and the current system of PNAS were you learn the identity of the editor only if your paper is accepted and after is published, far superior (you make friends but not enemies ...) I would not mind to had seen IUCr journals adopting a similar system, I think it would improve even further their good reputation. A. On Nov 19, 2010, at 12:32, John R Helliwell wrote: I don't wish to vear away from Victor's thrust with starting this thread and I would happily sign the petition you suggest. But I feel I should respond to the assertions about 'problems of peer review' at least with respect to Journals of my experience. Some 'Editor handling of submissions' statistics should help quantify such matters. These are a matter of public record re my IUCr Journals submission handling statistics ie therefore not confidential and which basically are:- approx 1000 article submissions; my rejection rate 20%; appeals against my rejections 0.5%; As Editor in Chief of Acta Cryst between 1996 to 2005 I received three appeals (out of approx tens of thousands of submissions through all Coeditors); I rejected these three. [My judgements were confidential re the details.] I can add that for the 2000 referees' reports or so for my article handling of submissions, that colleagues have kindly supplied to my Editor requests, problems involve:- about 1% where the report is 'publish as is' AND without any commendation given; these are in effect not terribly useful reports to me as an Editor. Another problem, which is growing, is the number of declines to my invites to referee (around 10%). Even worse are the no replies at all from invited referees as time is lost to the authors who rightly expect as prompt as possible handling. Re your points I offer replies as follows:- Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement takes a temporary leave of abesnse. JRH reply:- Such reports would be easy to spot and are not a problem in my experience and so resort to double blind review is not necessary in my experience. 2. 'preferred reviewers'. a double edged sword .. think about it. JRH reply; these are not so commonly offered suggestions by authors in fact and where they are one can follow or decide against (see point 1). 3. too much power of decision on editors (professional or academic) being able to reject papers without peer-review in many journals. JRH reply;This approach, 'insufficent general interest' is for the magazines we know and yet still love. 4. Bad refereeing - sometimes I wonder if people read the paper. JRH reply;Such reports are very few and obvious. The other categories above are more common (ie 'publish as is' category). 5. Lack of referee expertise: you get papers these days with: a structure, some biochemistry, some SAXS, some biophysics, and a cell based assay. Two or three people being able to pick up all the mistakes is very unlikely. JRH reply; Papers can be challenging re content and your example here is a good one. Other chalenging cases are where they include a lot of maths. That said peer review does its best but can occasionally fail; this level of failure can be measured by the number of criticism articles or formal retractions. These are also very few, but it is true, not zero. Yours sincerely, John On Thu, Nov 18, 2010 at 10:47 AM, Anastassis Perrakis a.perra...@nki.nl wrote: On Nov 18, 2010, at 11:18, James Stroud wrote: The future of publishing will be (1) Publish your own work (2) Peer review by the entire community Although I have been remarkably bad at predicting the future, I still like attempting to do so ...! This will not happen ...! ;-) To be honest, I am not even sure its a great idea ... Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement
Re: [ccp4bb] origin_shift in polar space group
Where do I find documentation for csymmatch ? Google is normally good at finding program documentation (and sometimes code which is infinitely better) but not in this case - I even tried spelling it 'csymatch' just in case! I was just interested to know whether csymmatch tries all combinations of matching A to A, A to B, A to C, B to C etc etc.? That's what people usually forget to do - i.e. they fail to observe the obvious that NCS-related molecules are not created identical! -- Ian On Fri, Nov 19, 2010 at 11:45 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote: Have you tried csymmatch -pdbin-ref one.pdb -pdbin two.pdb That will move chains to match asfar as possible, using sym ops and allowedorigin shifts to generate the best fit. Eleanor On 11/18/2010 12:26 PM, Ian Tickle wrote: OK now I understand. I couldn't find the script 'origin.com' you mentioned in the examples directory (at least from the filename I assume it's a script, not a MS-DOS program!), but it doesn't matter, I see the problem now. AFAIK there isn't a script in CCP4 that will do what you want entirely automatically, because it's actually quite a complicated problem in the completely general case of N molecules per a.u., though undoubtedly it could easily be scripted for the relatively simple case of 2 mols per a.u.. I'm assuming you don't simply want to superpose the molecules just for structural comparison purposes, you want to superpose the entire *crystals*, so that the calculated structure factors and hence the R factors (values) remained unchanged for the transformed structure. This means you can't use just arbitrary rotation/translation operators as would be generated by superposition programs such as SSM, you have to restrict it to crystallographically-allowed origin shifts. There are various programs which will do this, I wrote one called 'reforigin' but there are others which will do the same thing, and which have been mentioned in previous postings. So what you have to do is superpose the two 'A' molecules using reforigin or whatever (remember, as long as it applies only crystallographically-allowed origin shifts). There is of course a problem here: the chain ID 'A' is only an arbitrary label, so there's a 50% chance that the molecule you called 'A' in structure 1 might be called 'B' in structure 2 (and vice versa). This means you have to try both possibilities! Now you see why it gets complicated in the general case with molecules 'A', 'B', 'C', 'D' ... you have to try all combinations! While you are superposing A/2 on A/1 (or B/2 on A/1) you must also transform the other chain B/2 (or A/2) using the *same* operator (I think the program does this for you, or at least it will print the matrix that was used for the 1st pair) - you must not superpose it independently. Finally you need to transform the other molecule B/2 (or A/2) in the example above. For this you can only use space-group symmetry operators - you get only one chance to use the allowed origin shifts with the first pair of molecules, after that the origin is completely determined for the entire structure, hence only space-group symmetry can be used to transform subsequent pairs. For this I find it easiest just to view the structure on the graphics, work out which is appropriate space-group operator and apply it just to the 2nd molecule using PDBSET. Hope this is all clear - there are many traps here for the unwary! Cheers -- Ian On Thu, Nov 18, 2010 at 10:55 AM, Rojan Shrestharo...@riken.jp wrote: Hello Ian: I am afraid that whether my problem is not clear to you. Here is brief description of the problem. When I tried to superimpose two structures having two or more copies in ASU for polar space group using symmetric operator, for one copies it used one origin and for next, another origin is used. So there is origins shift problem. Here is an example: applying 0.50 0.50 -0.69 Y,-X,3/4+Z to chain A applying 1.50 0.50 0.61 X,Y,Z to chain B WARNING: ./input.pdb chain B is on a different origin! I used origin.com to superimpose two models. Now I hope you get the insight of my problem. Do you have any idea to solve this problem? Regards, Rojan -Original Message- From: Ian Tickle [mailto:ianj...@gmail.com] Sent: Thursday, November 18, 2010 7:42 PM To: ro...@riken.jp Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] origin_shift in polar space group HI Rojan, I'm not entirely clear that there is a problem. After superposition any origin shift that may have been present is removed: doesn't that solve your problem? Cheers -- Ian On Thu, Nov 18, 2010 at 10:11 AM, Rojan Shrestharo...@riken.jp wrote: Hello: In polar space group when the two or more copies molecules are superimposed, the origin is shifted. Does anybody have the solution to tackle this problem? Regards, Rojan
Re: [ccp4bb] Editorial policies (was Citations in supplementary material)
Regarding editorial decisions, I actually welcome editors making more rejection decisions, i.e. reading the paper before sending it to referees, so I waste less time, waiting as author, or as referee reading and commenting on papers for which it would have been clear beforehand that they are not suitable for the journal. I realise this would put more burden on the editors, but then they could enlist more editor (I have never been asked to be editor for any journal yet (hint)...). Mark Quoting Anastassis Perrakis: Dear John, I did not have the IUCr journals specifically in mind while making these remarks. Quite the contrary, I think due to you and other colleagues and friends, they are run competently and to the benefit of the community and of science at large. If I may though offer an opinion about the peer review system in IUCr journals, I personally find the concept that the authors know the identity of the managing editor, wrong. I am sure that in the majority of cases its not a problem, but often a managing editor can hesitate to communicate a negative referee report to e.g. an old colleague or good friend, whose manuscript she/he is handling, even when one of the referees is negative. I much prefer the system of e.g. Proteins (where I act as a managing editor), where authors never learn the identity of the managing editor far more comfortable (there is a few people that I would rather prefer if they don't know for sure that I rejected their paper), and the current system of PNAS were you learn the identity of the editor only if your paper is accepted and after is published, far superior (you make friends but not enemies ...) I would not mind to had seen IUCr journals adopting a similar system, I think it would improve even further their good reputation. A. On Nov 19, 2010, at 12:32, John R Helliwell wrote: I don't wish to vear away from Victor's thrust with starting this thread and I would happily sign the petition you suggest. But I feel I should respond to the assertions about 'problems of peer review' at least with respect to Journals of my experience. Some 'Editor handling of submissions' statistics should help quantify such matters. These are a matter of public record re my IUCr Journals submission handling statistics ie therefore not confidential and which basically are:- approx 1000 article submissions; my rejection rate 20%; appeals against my rejections 0.5%; As Editor in Chief of Acta Cryst between 1996 to 2005 I received three appeals (out of approx tens of thousands of submissions through all Coeditors); I rejected these three. [My judgements were confidential re the details.] I can add that for the 2000 referees' reports or so for my article handling of submissions, that colleagues have kindly supplied to my Editor requests, problems involve:- about 1% where the report is 'publish as is' AND without any commendation given; these are in effect not terribly useful reports to me as an Editor. Another problem, which is growing, is the number of declines to my invites to referee (around 10%). Even worse are the no replies at all from invited referees as time is lost to the authors who rightly expect as prompt as possible handling. Re your points I offer replies as follows:- Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement takes a temporary leave of abesnse. JRH reply:- Such reports would be easy to spot and are not a problem in my experience and so resort to double blind review is not necessary in my experience. 2. 'preferred reviewers'. a double edged sword .. think about it. JRH reply; these are not so commonly offered suggestions by authors in fact and where they are one can follow or decide against (see point 1). 3. too much power of decision on editors (professional or academic) being able to reject papers without peer-review in many journals. JRH reply;This approach, 'insufficent general interest' is for the magazines we know and yet still love. 4. Bad refereeing - sometimes I wonder if people read the paper. JRH reply;Such reports are very few and obvious. The other categories above are more common (ie 'publish as is' category). 5. Lack of referee expertise: you get papers these days with: a structure, some biochemistry, some SAXS, some biophysics, and a cell based assay. Two or three people being able to pick up all the mistakes is very unlikely. JRH reply; Papers can be challenging re content and your example here is a good one. Other chalenging cases are where they include a lot of maths. That said peer review does its best but can occasionally fail; this level of failure can be measured by the number of criticism articles or formal retractions. These are also very few, but it is true, not zero. Yours sincerely,
[ccp4bb] relationship between B factors and Koff
Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] Citations in supplementary material
Dear Tassos, Thankyou for sharing your own experiences as an Editor with Proteins, and with PNAS, which I really appreciate, and in particular for your suggestion of an anonymous Editor approach.This is interesting to me not least as one of the three Appeals I rejected, to which I referred below, led to what can only be described as 'hate email'. Indeed I will carry forward your suggestion to the IUCr Journals Commission via the Managing Editor (Peter Strickland), who I copy into this reply. [On a point of terminology within IUCr Journals we refer to the Editors and Co-Editors, who are scientific editors, and overall there is a Managing Editor based at IUCr HQ, which is Peter.] Yours sincerely, John cc On Fri, Nov 19, 2010 at 12:16 PM, Anastassis Perrakis a.perra...@nki.nl wrote: Dear John, I did not have the IUCr journals specifically in mind while making these remarks. Quite the contrary, I think due to you and other colleagues and friends, they are run competently and to the benefit of the community and of science at large. If I may though offer an opinion about the peer review system in IUCr journals, I personally find the concept that the authors know the identity of the managing editor, wrong. I am sure that in the majority of cases its not a problem, but often a managing editor can hesitate to communicate a negative referee report to e.g. an old colleague or good friend, whose manuscript she/he is handling, even when one of the referees is negative. I much prefer the system of e.g. Proteins (where I act as a managing editor), where authors never learn the identity of the managing editor far more comfortable (there is a few people that I would rather prefer if they don't know for sure that I rejected their paper), and the current system of PNAS were you learn the identity of the editor only if your paper is accepted and after is published, far superior (you make friends but not enemies ...) I would not mind to had seen IUCr journals adopting a similar system, I think it would improve even further their good reputation. A. On Nov 19, 2010, at 12:32, John R Helliwell wrote: I don't wish to vear away from Victor's thrust with starting this thread and I would happily sign the petition you suggest. But I feel I should respond to the assertions about 'problems of peer review' at least with respect to Journals of my experience. Some 'Editor handling of submissions' statistics should help quantify such matters. These are a matter of public record re my IUCr Journals submission handling statistics ie therefore not confidential and which basically are:- approx 1000 article submissions; my rejection rate 20%; appeals against my rejections 0.5%; As Editor in Chief of Acta Cryst between 1996 to 2005 I received three appeals (out of approx tens of thousands of submissions through all Coeditors); I rejected these three. [My judgements were confidential re the details.] I can add that for the 2000 referees' reports or so for my article handling of submissions, that colleagues have kindly supplied to my Editor requests, problems involve:- about 1% where the report is 'publish as is' AND without any commendation given; these are in effect not terribly useful reports to me as an Editor. Another problem, which is growing, is the number of declines to my invites to referee (around 10%). Even worse are the no replies at all from invited referees as time is lost to the authors who rightly expect as prompt as possible handling. Re your points I offer replies as follows:- Let me outline what I think are problems of peer review: 1. 'review by last author name'. Very often the last author is well known, or a friend, and the reviewers' critical judgement takes a temporary leave of abesnse. JRH reply:- Such reports would be easy to spot and are not a problem in my experience and so resort to double blind review is not necessary in my experience. 2. 'preferred reviewers'. a double edged sword .. think about it. JRH reply; these are not so commonly offered suggestions by authors in fact and where they are one can follow or decide against (see point 1). 3. too much power of decision on editors (professional or academic) being able to reject papers without peer-review in many journals. JRH reply;This approach, 'insufficent general interest' is for the magazines we know and yet still love. 4. Bad refereeing - sometimes I wonder if people read the paper. JRH reply;Such reports are very few and obvious. The other categories above are more common (ie 'publish as is' category). 5. Lack of referee expertise: you get papers these days with: a structure, some biochemistry, some SAXS, some biophysics, and a cell based assay. Two or three people being able to pick up all the mistakes is very unlikely. JRH reply; Papers can be challenging re content and your example here is a good one. Other chalenging cases are where they include a lot of maths.
Re: [ccp4bb] relationship between B factors and Koff
I can direct you to PDB entry 1EWY, where the average isotropic temperature factor for the major component of the complex is ca. 47 A**2 and that for the smaller component is ca. 69 A**2. Similar values than the ones you are reporting. I am assuming some sort of disorder, or if you prefer, wobbling of the smaller component at the lever of the binding site. Fred. Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
[ccp4bb] nVidia stereo problems : Mac Pro, Quadro FX 4800
(Apologies to those who have already seen this on the PyMol list) Does anyone have experience of a working Mac Pro/Quadro FX 4800/120Hz flat screen setup? I'm trying to put one together, and am getting nowhere. We have: - Mac Pro (early 2010, MacPro 5,1) running 10.6.4 - Quadro FX 4800 for Mac - nVidia drivers Retail_256.01.00f03 (latest as of 19/10/2010) - nVidia IR emitter and glasses set connected by DIN and USB cables - ViewSonic Fuhzion VX2268WM, refresh 120Hz We have working Linux setups that are the same as this (apart from the Mac Pro), and I've tested that the graphics card, monitor, glasses and emitter all work in Stereo. With the Mac, I've tried both Stereo-capable native apps (PyMol incentive product, QtMG) and X11 apps (Coot). The problem is that the emitter refuses to turn on; it just sits there flashing red. The display looks as it should for stereo. nVidia's support for Mac is effectively non-existent, so I'm hoping that someone on ccp4bb has got this working in the past and knows the correct incantations. Regards, Chris -- Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] relationship between B factors and Koff
A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much
Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano, I don't see how the k-off would influence this, given the timescale of growing crystals. An explanation in terms of high Kd and relative lack of crystal contacts for the component with higher temperature factors would sound more convincing to me. Mark Quoting Vellieux Frederic: I can direct you to PDB entry 1EWY, where the average isotropic temperature factor for the major component of the complex is ca. 47 A**2 and that for the smaller component is ca. 69 A**2. Similar values than the ones you are reporting. I am assuming some sort of disorder, or if you prefer, wobbling of the smaller component at the lever of the binding site. Fred. Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s 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] relationship between B factors and Koff
I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help here). Good luck and cheers~ ~Justin Quoting Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on
Re: [ccp4bb] relationship between B factors and Koff
The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end concentration. A colleague of mine once told me this is due to a 5 to 10-fold dilution effect upon addition to the column, but I have never verified this nor read any primary source that validated this so I cannot supply a reference (others might be able to help
Re: [ccp4bb] relationship between B factors and Koff
Well, if one of the components is a small peptide and the solutions are dilute, it is hard to concentrate the complex, as the free peptide will go through the concentrator! It's a tricky problem. JPK On Fri, Nov 19, 2010 at 9:38 AM, herman.schreu...@sanofi-aventis.com wrote: The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might be tempting for you to draw this comparison in your structure, I would warn that this is too large a leap without further (non-anecdotal) evidence. As a further note, during SEC purification of complexes, I have observed that you generally have to have the complexes at at least 5 to 10-fold higher initial concentration if you want to purify the complex, which you are only pushing with your 80-100 uM high end
Re: [ccp4bb] relationship between B factors and Koff
If the peptide is really small compared to the protein, I would just add excess peptide and not worry about the stochiometry. Best, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 5:03 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff Well, if one of the components is a small peptide and the solutions are dilute, it is hard to concentrate the complex, as the free peptide will go through the concentrator! It's a tricky problem. JPK On Fri, Nov 19, 2010 at 9:38 AM, herman.schreu...@sanofi-aventis.com wrote: The amount of small component one looses from the complex depends on the Koff and the local concentrations during the SEC run, so I doubt if one could estimate those losses with an error less than the 1% pipetting error you quote. What I would do is to pipet dilute solutions, and concentrate afterwards. If there are conditions which give bad crystals, one could also harvest these crystals, redissolve them and set up crystallizations with those. My two cents, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Friday, November 19, 2010 3:57 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] relationship between B factors and Koff I should add that this procedure is really only advantageous for high Koff complexes. If the complex does not dissociate appreciably in the time required for SEC, I agree that there is no great benefit for doing it my way. I have been working recently, however, on a high Koff complex, so have been thinking about how to get exactly the right ratio (other suggestions welcome!) Jacob On Fri, Nov 19, 2010 at 8:45 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, mixing together has a much bigger pipetting error (to get, say, 500uM, you have to add 1000uM proteins A and B together, so with a pipetting error of ~1% even (and concentrated protein solutions seem to be tricky to pipette accurately), there would be an error of 10uM. Also, there are the errors associated with concentration determination, which are probably not trivial, especially with low EC. If, however, one of the components A is preloaded at low concentration (1-5uM, say), as I have recommended, the excess of that component with be exactly 1-5uM, assuming the complex was loaded with a slight excess of A. And this, as a percentage of the total 500uM, is much less than the various errors involved in mixing the two together. So this would be the procedure: Mix A with B at a calculated stoichiometric ratio of 1.1:1.0 Preload the column with a low level of A just before injecting the complex Inject the complex Collect fractions, solve structure, publish in your favorite venue JPK On Fri, Nov 19, 2010 at 8:30 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Jacob, The SEC is generally run to separate the complex from the unbound components. If run the way your propose, the peak of unbound preinjected smaller component coincides with the peak of the complex and the final stochiometry is not better than by just mixing the components without SEC. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, November 19, 2010 3:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] relationship between B factors and Koff A suggestion for purifying the complex: let's say there is a 5mL gap between the complex and one of its (smaller)constituents A. You can pre-load the column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to be injected right after the pre-load. This should provide approximately equilibrium conditions, so that the complex should be basically 1:1 when it comes out, even with a high Koff. (Alternatively, for true equilibrium conditions, just equilibrate the entire column in A, then inject the complex.) JPK - Original Message - From: Justin Hall hallj...@onid.orst.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 7:32 AM Subject: Re: [ccp4bb] relationship between B factors and Koff Hi Sebastiano, I have had some experience with protein:protein complexes with KD ~ 10-1 uM, kinetic characterization and trying to purify a complex of these proteins using SEC. While I would say that if you have reliable evidence from SPR that you have a fast on (high Kon), then you must have a fast off (high Koff) because by definition KD = 10 E-6 = Koff/Kon. However, I have observed several systems where you have a KD ~ 10-1 uM, but the kinetics are not fast on/fast off. In my experience, I have never seen anything in the crystal structures of the weak affinity complexes I have solved that would coorelate B-factors to Kon/Koff, and while it might
Re: [ccp4bb] relationship between B factors and Koff
This doesn't directly address your question, but since the subject of analyzing protein-protein interactions with gel filtration is raised on this bb occasionally, I thought I would mention that there are cases in which conventional gel filtration chromatography fails to provide evidence of a known protein:protein interaction. In such cases, the Hummel-Dreyer gel filtration method is sometimes used. It involves supplementing the running buffer with one of the proteins, so you need a lot of protein. Here are two references: Proc. Natl. Acad. Sci. USA 88 (1991) Biochemisrry (1993) 32, 11124-11131 On 11/19/10 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] relationship between B factors and Koff
Yes, this is where I heard of the technique, and my suggestion was a modification of that, but my references were: Hummel JP Dreyer WJ (1962) Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta 63:530-532. Ratner D (1974) The interaction bacterial and phage proteins with immobilized Escherichia coli RNA polymerase. J Mol Biol 88(2):373-383. JPK - Original Message - From: Tanner, John J. tanne...@missouri.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, November 19, 2010 11:25 AM Subject: Re: [ccp4bb] relationship between B factors and Koff This doesn't directly address your question, but since the subject of analyzing protein-protein interactions with gel filtration is raised on this bb occasionally, I thought I would mention that there are cases in which conventional gel filtration chromatography fails to provide evidence of a known protein:protein interaction. In such cases, the Hummel-Dreyer gel filtration method is sometimes used. It involves supplementing the running buffer with one of the proteins, so you need a lot of protein. Here are two references: Proc. Natl. Acad. Sci. USA 88 (1991) Biochemisrry (1993) 32, 11124-11131 On 11/19/10 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990 *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] relationship between B factors and Koff
Sebastiano, We observed similar phenomenon in our protein-protein complex. This reference will give you details (Crystal Structure of an Electron Transfer Complex between Aromatic Amine Dehydrogenase- Azurin from Alcaligenes faecalis, Biochemistry, 45, 13500-13510, 2006). Sukumar Narayanasami Sukumar NE-CAT, Building 436E Advanced Photon Source Argonne National Laboratory 9700 S.Cass Ave Argonne, IL 60439-4800 USA Tel: 630-252-0681 Fax: 630-252-0687 e-mail: suku...@aps.anl.gov On Fri, Nov 19, 2010 at 6:58 AM, Sebastiano Pasqualato sebastiano.pasqual...@ifom-ieo-campus.it wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094 fax +39 02 9437 5990
Re: [ccp4bb] Citations in supplementary material
I took a look at his slides--looks pretty interesting, and I would have liked to have heard the talk! I am not sure I would be ready to advocate having no more journals, but I am interested in thinking about the idea. Imagine just putting your data up on the website, and you're finished! And then wait for comments/links to show up, I guess--would that be the metric for grants/funding? I also wonder what could be done so that the record would not be changed? JPK On Thu, Nov 18, 2010 at 3:14 AM, John R Helliwell jrhelliw...@gmail.com wrote: Dear Jacob, Your posting reminds me of a Research Information Network Conference I went to in 2006 in London. Your views coincide with a presenter there, Peter Mika. His talk can be found at:- http://www.rin.ac.uk/news/events/data-webs-new-visions-research-data-web In his talk he referred to:- openacademia.org Peter Mika and I were on the Closing Panel; he advocated that refereeing is an imposition on a researcher's individual freedom and thus he/she should 'publish' their work on their own website. By contrast, I argued in favour of Journals and peer review, both with respect to my articles and my experiences as an Editor of more than one Journal. I would be happy to continue corresponding on this not least as publication should be a varied spectrum of options. Also I feel obliged to say that one cannot apply simply, by rote, 'Learned Society publisher is good', 'commercial publisher is bad'; there are exceptions in both camps. [in effect this was the tone of my last posting.] Greetings, John On Wed, Nov 17, 2010 at 8:13 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I guess the practice of being on your best behavior is good in terms of getting the research trimmed into shape, but there is a huge temptation to fudge things to get published, and to hide unpleasant artifacts, as can be seen by the many recent (and not so recent) scandals. Maybe as a lab website things would be more open. Also, having a comments section always seemed like an excellent idea to me, even for journals as they are, but would be really easy to implement in a website. I would love to read comments from others in the field about the papers I read, as sometimes people can help to point out gaping holes where one might not see them otherwise. It would be like journal club for the whole scientific community. Jacob On Wed, Nov 17, 2010 at 2:08 PM, Jrh jrhelliw...@gmail.com wrote: Dear Jacob Re journals out of the window:- Well, like democracy, journals may not be ideal but I believe other alternatives such as free for all personal website publishing, are worse. So, journals that are community driven offer an optimal approach, critically based on specialist peer review. That is why our community effort IUCr Journals I believe are so important. Open access, where we can sustain it financially, also can convey access to the widest readership ie that the high impact magazines currently, mainly, command. All best wishes, John Prof John R Helliwell DSc On 17 Nov 2010, at 18:28, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Supplementary info seems to me to be a double-edged sword--I just read a Nature article that had 45 pages of supplementary info. This means that you get a lot more for your money, but all of the methods and Why not have papers be as long as the authors want, now that almost everything is internet-based? It would make the papers much more organized overall, and would obviate the reference issue mentioned in this thread. To avoid them being too too long, reviewers could object to long-windedness etc. But, it would definitely make for a more complete lab notebook of the scientific community, assuming that that is what we are after. Incidentally, I have been curious in the past why journals are not going out the window themselves--why not have individual labs just post their most recent data and interpretations on their own websites, with a comments section perhaps? (I know there are about a thousand cynical reasons why not...) One could even have a place for reliability rating or impact rating on each new chunk of data. Anyway, it would be much more like a real-time, public lab notebook, and would make interaction much faster, and cut out the publishing middlemen. JPK On Wed, Nov 17, 2010 at 11:48 AM, Phoebe Rice pr...@uchicago.edu wrote: Another unfortunate aspect of this sort of editorial policy is that many of these papers contain almost no technical information at all, except for the supplement. I've started to avoid using Nature papers for class discussions becuase they leave the students so puzzled, and with a glossiness-is-all-that-matters idea of science. = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723
Re: [ccp4bb] Citations in supplementary material
On Fri, Nov 19, 2010 at 11:33 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I took a look at his slides--looks pretty interesting, and I would have liked to have heard the talk! I am not sure I would be ready to advocate having no more journals, but I am interested in thinking about the idea. Imagine just putting your data up on the website, and you're finished! And then wait for comments/links to show up, I guess--would that be the metric for grants/funding? I also wonder what could be done so that the record would not be changed? You can't rely on personal academic websites for long-term archival of anything. This is one reason why institutions such as the PDB and GenBank (among others) exist, to ensure that published data is accessible at a central location, and there is considerable economy of scale from doing it this way. What's missing, of course, is any associated content like you propose, but organizations like the structural genomics groups (at least in the US) are already halfway there, since they immediately release their structures, presumably before they even write a paper. Large genomics centers do the same with their raw sequencing data. However, structural genomics is a very different enterprise from traditional hypothesis-driven science, and I'm not convinced that the immediate-release model will ever be applicable to most academics. Anyone with sufficient technical skill and proper resources can generate raw data; figuring out what it means, and what follow-up experiments need to be done, is much harder. There are many ways to incorporate collaborative annotation and other unconventional methods of publication; I think PLoS ONE (which allows comments) is an excellent step in the right direction. However, until we start seeing ribosome or membrane-protein structures published this way, everyone with an eye on a future academic career is going to prefer the traditional route. -Nat
Re: [ccp4bb] Citations in supplementary material
I think you are right--it would have to be a centralized site, like the openacademia.org proposed by Peter Mika. I think there should be several levels in the site, which would correspond to level of closeness to phenomena, somewhat like the progression from abstract discussion results methods. I guess since science is trying to build a bridge between the inner truths of the mind and the phenomena of the senses, the structure of scientific writing has worked out that way. Probably that should be preserved in the website. JPK On Fri, Nov 19, 2010 at 2:05 PM, Nat Echols nathaniel.ech...@gmail.com wrote: On Fri, Nov 19, 2010 at 11:33 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I took a look at his slides--looks pretty interesting, and I would have liked to have heard the talk! I am not sure I would be ready to advocate having no more journals, but I am interested in thinking about the idea. Imagine just putting your data up on the website, and you're finished! And then wait for comments/links to show up, I guess--would that be the metric for grants/funding? I also wonder what could be done so that the record would not be changed? You can't rely on personal academic websites for long-term archival of anything. This is one reason why institutions such as the PDB and GenBank (among others) exist, to ensure that published data is accessible at a central location, and there is considerable economy of scale from doing it this way. What's missing, of course, is any associated content like you propose, but organizations like the structural genomics groups (at least in the US) are already halfway there, since they immediately release their structures, presumably before they even write a paper. Large genomics centers do the same with their raw sequencing data. However, structural genomics is a very different enterprise from traditional hypothesis-driven science, and I'm not convinced that the immediate-release model will ever be applicable to most academics. Anyone with sufficient technical skill and proper resources can generate raw data; figuring out what it means, and what follow-up experiments need to be done, is much harder. There are many ways to incorporate collaborative annotation and other unconventional methods of publication; I think PLoS ONE (which allows comments) is an excellent step in the right direction. However, until we start seeing ribosome or membrane-protein structures published this way, everyone with an eye on a future academic career is going to prefer the traditional route. -Nat
Re: [ccp4bb] relationship between B factors and Koff
I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
Maybe you should look at: Rotation barriers in crystals from atomic displacement parameters Emily Maverick and Jack Dunitz (1987) Vol 62(2), 451-459. They reported that the libration amplitude of one part of a crystalline small molecule relative to another part is strongly correlated to bond rotation barriers measured by other means. The discussion at the top of page 458 seems to be about off rates. It's not up to me if a multi-temperature diffraction experiment and a lot of TLS analyses will be worth the effort relative to purchasing equipment to measure a binding parameter. -Dan James Holton wrote: I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
Re: [ccp4bb] relationship between B factors and Koff
The journal was Molecular Physics (1987) Vol 62(2), 451-459. (my brain librates when I type) Daniel Anderson wrote: Maybe you should look at: Rotation barriers in crystals from atomic displacement parameters Emily Maverick and Jack Dunitz (1987) Vol 62(2), 451-459. They reported that the libration amplitude of one part of a crystalline small molecule relative to another part is strongly correlated to bond rotation barriers measured by other means. The discussion at the top of page 458 seems to be about off rates. It's not up to me if a multi-temperature diffraction experiment and a lot of TLS analyses will be worth the effort relative to purchasing equipment to measure a binding parameter. -Dan James Holton wrote: I don't think there is any relationship between rate constants and B factors. Yes, there is the hand-wavy argument of disorder begets disorder (and people almost always LITERALLY wave their hands when they propose this), but you have to be much more careful than that when it comes to thermodynamics. Yes, disorder and entropy are related, but just because a ligand is disordered does not mean that the delta-S term of the binding delta-G is higher. Now, there is a relationship between equilibrium constants and occupancies, since the occupancy is really just the ratio of the concentration of protein-bound-to-ligand to the total protein concentration, and an equilibrium exists between these two species. NB all the usual caveats of how crystal packing could change binding constants, etc. You could logically extend this to B factors by invoking a property of refinement: Specifically, if the true occupancy is less than 1, but modeled as 1.00, your refinement program will give you a B factor that is larger than the true atomic B factor. However, if you try to make this claim, then the obvious cantankerous reviewer suggestion would be to refine the occupancy. Problem is, refining both occupancy and B at the same time is usually unstable at moderate resolution. In general, it is hard to distinguish between something that is flopping around (high B factor) and something that is simply not there part of the time (low occupancy). I know it is tempting to try and relate B factors directly to entropy, but the disorder that leads to large B factors has a lot more to do with crystals than it has to do with proteins. For example, there are plenty of tightly-bound complexes that don't diffract well at all. If you refine these structures, you will get big B factors (roughly, B = 4*d^2+12 where d is the resolution in Angstrom). You may even have several crystal forms of the same thing with different Wilson B factors, but that is in no way evidence that the proteins in the two crystal forms somehow have different binding constants or rate constants. On the bright side, in your case it sounds like you have an entire protomer that is disorered relative to the rest of the crystal lattice. We have seen a few cases now like this where dehydrating the crystal (with an FMS or similar procedure) causes the unit cell to shrink and this locks the wobbly molecule into place. I think this is the principle mechanism of improved diffraction from dehydration. No, it does not work very often! But sometimes it does. -James Holton MAD Scientist On 11/19/2010 4:58 AM, Sebastiano Pasqualato wrote: Hi all, I have a crystallographical/biochemical problem, and maybe some of you guys can help me out. We have recently crystallized a protein:protein complex, whose Kd has been measured being ca. 10 uM (both by fluorescence polarization and surface plasmon resonance). Despite the 'decent' affinity, we couldn't purify an homogeneous complex in size exclusion chromatography, even mixing the protein at concentrations up to 80-100 uM each. We explained this behavior by assuming that extremely high Kon/Koff values combine to give this 10 uM affinity, and the high Koff value would account for the dissociation going on during size exclusion chromatography. We have partial evidence for this from the SPR curves, although we haven't actually measured the Kon/Koff values. We eventually managed to solve the crystal structure of the complex by mixing the two proteins (we had to add an excess of one of them to get good diffraction data). Once solved the structure (which makes perfect biological sense and has been validated), we get mean B factors for one of the component (the larger) much lower than those of the other component (the smaller one, which we had in excess). We're talking about 48 Å^2 vs. 75 Å^2. I was wondering if anybody has had some similar cases, or has any hint on the possible relationship it might (or might not) exist between high a Koff value and high B factors (a relationship we are tempted to draw). Thanks in advance, best regards, ciao s
[ccp4bb] per-residue RMSD calculation for homologous structure
Hi All does anyone know any software that can calculate and print out RMSD of every residue (c alpha will be good) for homologous structures which has only 30-40 % sequence similarity? I looked on the web but all the software that I found require the sequence to be the same for both structure. Thank you Dhiraj
Re: [ccp4bb] per-residue RMSD calculation for homologous structure
On Friday, November 19, 2010 02:55:54 pm Srivastava, Dhiraj (MU-Student) wrote: Hi All does anyone know any software that can calculate and print out RMSD of every residue (c alpha will be good) for homologous structures which has only 30-40 % sequence similarity? I looked on the web but all the software that I found require the sequence to be the same for both structure. This is not a well-defined calculation. In regions where the structures do not superimpose well, how do you decide which C-alpha pairs with which? What about insertions/deletions? Usually it only makes sense to calculate RMSD over those residues which line up structurally, so it won't be every residue. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] per-residue RMSD calculation for homologous structure
I looked on the web but all the software that I found require the sequence to be the same for both structure. most modern programs perform some kind of local-global alignment first to define the corresponding residues and then compute the statistics. BR
Re: [ccp4bb] per-residue RMSD calculation for homologous structure
Dear Dhiraj; Please try the following web site http://www.cgl.ucsf.edu/home/meng/grpmt/structalign.html Here you will find a number of option for structure base sequence alignment no matter what is the similarity of your structures. Regarding the RMSD of every residues you can find this option by using the Dalilite server. Good luck Bashir On Fri, November 19, 2010 23:55, Srivastava, Dhiraj (MU-Student) wrote: Hi All does anyone know any software that can calculate and print out RMSD of every residue (c alpha will be good) for homologous structures which has only 30-40 % sequence similarity? I looked on the web but all the software that I found require the sequence to be the same for both structure. Thank you Dhiraj -- Muhammad Bashir Khan ** Department for Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria Austria Phone: +43(1)427752224 Fax: +43(1)42779522
Re: [ccp4bb] Off Topic - Nickel Column
Depending on the type of resin you should be able to use IMAC all the way up to pH 11 or so (works for Ni-NTA, not sure about HIS-SELECT or other resins). Obviously your buffer system changes (carbonate works well at 11). Why not to elute the complex together with pure monomer and then try separation via SEC or some other means? Or flush the column with a gradient of urea or guanidine to see where the remaining partner protein elutes? Notably, if your interaction is hydrophobic in nature, high salt will strengthen it. By the sound of things, the remaining 20% is probably a part of a semi-denatured aggregate, so a detergent, organic, or chaotrope wash is likely to help. Artem On Wed, Nov 17, 2010 at 10:49 AM, Daniel Bonsor bon...@bbri.org wrote: I have a His-tagged protein which I am coexpressing with it's binding partner to prevent proteolysis. Once on the Nickel column I can remove 80% of the partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight. However the last 20% is difficult to remove, even if I reload the Nickel column and flush a further 2l of salt solution. I am wondering if I can increase the pH to 9.0 or 9.5. It should not effect the binding of His for the Nickel as the His-tag has to be deprotonated to bind, though will it causing stripping of the Nickel? Thanks Dan
Re: [ccp4bb] - subtracting diffraction images?
On 10:51 Wed 17 Nov , Warren, Mark R wrote: I am trying to compare the difference between two diffraction images from a Mar detector before and after irradiation. At present I have used the marcombine program to subtract the diffraction images, but I don't have any statistical information. Can anyone suggest either another good program to observe the differences in the images or a way to obtain some statistics about the subtracted image? ADXV will allow you to treat one image as background and thus subtract it from another. There are no statistics, but you can also use ADXV to export a full-size image to TIFF and analyze it using more capable image-analysis tools like ImageJ. Of course a more advanced analysis would probably involve indexing each image and comparing intensities, but that's pretty straightforward so I'm assuming you've already thought about it. -- Thanks, Donnie Donald S. Berkholz, Ph.D. Research Fellow James R. Thompson lab, Physiology Biomedical Engineering Grazia Isaya lab, Pediatric Adolescent Medicine Medical Sciences 2-66 Mayo Clinic College of Medicine 200 First Street SW Rochester, MN 55905 office: 507-538-6924 cell: 612-991-1321 pgpZdedeRpFf9.pgp Description: PGP signature
Re: [ccp4bb] Off Topic - Nickel Column
I wish to thank everyone. I did try shifting to a higher pH and flushing 3l of salt solution over the column which did not work. I tried 20ml 2M Urea on the column and a stepwise shift to no urea that showed removal of the protein. I will try binding studies to see if I did not denature the His-tagged protein. Thanks for your suggestions. Dan
