Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) [SEC=UNCLASSIFIED]
I reckon you could share hypothetical review comments for educational purposes. -Original Message- From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thu 10/28/2010 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Why not double open review? If I have something reasonable to say, I should be able to sign it. Particularly if the publicly purported point of review is to make the manuscript better. And imagine what wonderful open hostility we would enjoy instead of all these hidden grudges! You would never have to preemptively condemn a paper on grounds of suspicion that it is from someone who might have reviewed you equally loathful earlier. You actually know that you are creaming the right bastard! A more serious question for the editors amongst us: Can I publish review comments or are they covered under some confidentiality rule? Some of these gems are quite worthy public entertainment. Best, BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Wednesday, October 27, 2010 6:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) What about the possibility of double-blind review? I have actually wondered why the reviewers should be given the author info--does that determine the quality of the work? Am I missing some obvious reason why reviewers should know who the authors are? JPK On Wed, Oct 27, 2010 at 5:50 PM, Phoebe Rice pr...@uchicago.edu wrote: Journal editors need to know when the reviewer they trusted is completely out to lunch. So please don't just silently knuckle under! It may make no difference for Nature, but my impression has been that rigorous journals like JMB do care about review quality. Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty _Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Wed, 27 Oct 2010 15:13:03 -0700 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com) Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) To: CCP4BB@JISCMAIL.AC.UK Surely the best model is the one that the referees for your paper are happy with? That may be the sad and pragmatic wisdom, but certainly not a truth we should accept... I have found referees to impose seemingly random and arbitrary standards a) Reviewers are people belonging to a certain population, characterized by say a property 'review quality' that follows a certain distribution. Irrespective of the actual shape of that parent distribution, the central limit theorem informs us that if you sample this distribution reasonably often, the sampling distribution will be normal. That means, that half of the reviews will be below average review quality, and half above. Unfortunately, the mean of that distribution is b) a function of journal editor quality (they pick the reviewers after all) and c) affected by systematic errors such as your reputation and the chance that you yourself might sit on a reviewer's grant review panel By combining a, b, c you can get a fairly good assessment of the joint probability of what report you will receive. You do notice that model quality is not a parameter in this model, because we can neglect marginal second order contributions. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. I agree and sympathize with your optimism, but I am afraid that those who might need this education are not the ones who seek it. I.e., reading the bb complicates matters (simplicity being one benefit of ROTs) and you can't build an empire wasting time on such things. Good luck with your reviews! BR Simon On 27 Oct 2010, at 20:11, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Young and Impressionable readers: I second-guess here that Robbie's intent - after re-refining many many PDB structures, seeing dreadful things, and becoming a hardened cynic - is to provoke more discussion in order to put in perspective - if not debunk- almost all of these rules. So it may be better to pretend you have never heard of these rules. Your crystallographic life might be a happier and less biased one. If you follow this simple procedure (not a rule) The model that fits the primary evidence (minimally biased electron density) best and is at the same time physically meaningful, is the best model, i. e., all plausibly accountable electron density (and not more) is modeled. This process of course does require a little work (like looking
Re: [ccp4bb] Against Method (R)
It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives similar results to R(sigma), both attempt to measure the precision of the MERGED data, which are what one is refining against. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 27 Oct 2010, Ed Pozharski wrote: On Tue, 2010-10-26 at 21:16 +0100, Frank von Delft wrote: the errors in our measurements apparently have no bearing whatsoever on the errors in our models This would mean there is no point trying to get better crystals, right? Or am I also wrong to assume that the dataset with higher I/sigma in the highest resolution shell will give me a better model? On a related point - why is Rmerge considered to be the limiting value for the R? Isn't Rmerge a poorly defined measure itself that deteriorates at least in some circumstances (e.g. increased redundancy)? Specifically, shouldn't ideal R approximate 0.5*sigmaI/I? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the best model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little to no impact on the biology being described. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. Simon On 27 Oct 2010, at 20:11, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Young and Impressionable readers: I second-guess here that Robbie's intent - after re-refining many many PDB structures, seeing dreadful things, and becoming a hardened cynic - is to provoke more discussion in order to put in perspective - if not debunk- almost all of these rules. So it may be better to pretend you have never heard of these rules. Your crystallographic life might be a happier and less biased one. If you follow this simple procedure (not a rule) The model that fits the primary evidence (minimally biased electron density) best and is at the same time physically meaningful, is the best model, i. e., all plausibly accountable electron density (and not more) is modeled. This process of course does require a little work (like looking through all of the model, not just the interesting parts, and thinking what makes sense) but may lead to additional and unexpected insights. And in almost all cases, you will get a model with plausible statistics, without any reliance on rules. For some decisions regarding global parameterizations you have to apply more sophisticated test such as Ethan pointed out (HR tests) or Ian uses (LL-tests). And once you know how to do that, you do not need any rules of thumb anyhow. So I opt for a formal burial of these rules of thumb and a toast to evidence and plausibility. And, as Gerard B said in other words so nicely: Si tacuisses, philosophus mansisses. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robbie Joosten Sent: Tuesday, October 26, 2010 10:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Dear Anthony, That is an excellent question! I believe there are quite a lot of 'rules of thumb' going around. Some of them seem to lead to very dogmatic thinking and have caused (refereeing) trouble for good structures and lack of trouble for bad structures. A lot of them were discussed at the CCP4BB so it may be nice to try to list them all. Rule 1: If Rwork 20%, you are done. Rule 2: If R-free - Rwork 5%, your structure is wrong. Rule 3: At resolution X, the bond length rmsd should be than Y (What is the rmsd thing people keep talking about?) Rule 4: If your resolution is lower than X, you should not use_anisotropic_Bs/riding_hydrogens Rule 5: You should not build waters/alternates at resolutions lower than X Rule 6: You should do the final refinement with ALL reflections Rule 7: No one cares about getting the carbohydrates right Obviously, this list is not complete. I may also have overstated some of the rules to get the discussion going. Any addidtions are welcome. Cheers, Robbie Joosten Netherlands Cancer Institute Apologies if I have missed a recent relevant thread, but are lists of rules of thumb for model building and refinement? Anthony Anthony Duff Telephone: 02 9717 3493 Mob: 043 189 1076 =
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
I do not know if that's really cynical: I've had the case of a referee recommending manuscript rejection because the title of the manuscript was not appropriate. The editor followed the advice of the referee. A proper refereeing job would have been to suggest that the authors change the title of the manuscript, not suggesting to the editor that the manuscript should be rejected! So I think we can have different opinions on this. Sometimes referees do a good job in evaluating manuscripts, sometimes they do not. Fred. Eleanor Dodson wrote: Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the best model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little to no impact on the biology being described. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. Simon
[ccp4bb] Additional band on gel due to his-tag: any references?
Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de
Re: [ccp4bb] Bug in c_truncate?
Dear Peter, it seems to me that you are having trouble with f2mtz and not with ctruncate, so I am confused by the subject. Can you please post - the error message, - the first couple of lines of the hkl-file you are trying to import (including one or two reflections which are flagged for Rfree), - the version of ccp4 you are using - whether you are doing the conversion from the GUI or the command line - if the latter, please also post the script you are using. Cheers, Tim On Wed, Oct 27, 2010 at 09:14:36PM -0400, Peter Chan wrote: Hello, I've been struggling with F2MTZ and importing my hkl file into mtz by 'keeping existing freeR data'. I keep getting the error Problem with FREE column in input file. All flags apparently identical. Check input file. At the end of the day, it appears that this only happens in ctruncate and not in the old_truncate instead of ctruncate. Has anyone experienced a similar problem? Peter -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since referees know the names of the authors of the articles they are refereeing, you may need a lot more than a couple of references to appease them after you called them bloody-minded in a public email forum. My two cents, Tim On Thu, Oct 28, 2010 at 01:16:59PM +0200, Sebastiaan Werten wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Against Method (R)
So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:05 AM Subject: Re: [ccp4bb] Against Method (R) It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives similar results to R(sigma), both attempt to measure the precision of the MERGED data, which are what one is refining against. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 27 Oct 2010, Ed Pozharski wrote: On Tue, 2010-10-26 at 21:16 +0100, Frank von Delft wrote: the errors in our measurements apparently have no bearing whatsoever on the errors in our models This would mean there is no point trying to get better crystals, right? Or am I also wrong to assume that the dataset with higher I/sigma in the highest resolution shell will give me a better model? On a related point - why is Rmerge considered to be the limiting value for the R? Isn't Rmerge a poorly defined measure itself that deteriorates at least in some circumstances (e.g. increased redundancy)? Specifically, shouldn't ideal R approximate 0.5*sigmaI/I? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs *** 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] Additional band on gel due to his-tag: any references?
Additional band on a gel might not be caused by his-tag. It is often a result of different conformation/molecular shape and so the molecule travels with different speed in the gel. We may wish for a homogenous sample (chemically and structurally) but this is seldom true See example: Int J Mol Med 2009, 23(1), 57 Jankun et al, VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form. I fully agree with Tim - making rude comments about reviewers (or anybody else) is not going to help you. Questioning an extra band is a legitimate remark that you should address. Good luck - Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave.e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Thursday, October 28, 2010 7:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Additional band on gel due to his-tag: any references? Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since referees know the names of the authors of the articles they are refereeing, you may need a lot more than a couple of references to appease them after you called them bloody-minded in a public email forum. My two cents, Tim On Thu, Oct 28, 2010 at 01:16:59PM +0200, Sebastiaan Werten wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] Bug in c_truncate?
Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried looking for known/resolved issues/updates in version 6.1.3 but could not find any so I assumed it is the same version of f2mtz/ctruncate/uniqueify. I used the GUI version of F2MTZ, with the settings below: - import file in SHELX format - keep existing FreeR flags - fortran format (3F4.0,2F8.3,F4.0) - added data label I other integer // FreeRflag The hkl file, in SHELX format, output by XPREP look something like this: -26 -3 1 777.48 39.19 26 -3 -1 800.83 36.31 -26 3 -1 782.67 37.97 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 Notice the test set is flagged -1 and the working set is not flagged at all. This actually lead to another error message in f2mtz about missing FreeR flags. From my understanding, the SHELX flagging convention is 1 for working and -1 for test. So I manually tagged the working set with 1 using vi: -26 -3 1 777.48 39.19 1 26 -3 -1 800.83 36.31 1 -26 3 -1 782.67 37.97 1 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 This is the file which gives me the error message: Problem with FREE column in input file. All flags apparently identical. Check input file.. Apparently, import to mtz works ok when I use old-truncate instead of c-truncate. Best, Peter
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
Hi Sebastian, Under the assumption that the SDS in your assay does not completely unfold the protein during electrophoresis (chemical impurity can be excluded because of MS experiments, right?), how about adding some urea additionally to the SDS-PAGE (or changing SDS concentration)? GL Jan --- Skrzypczak-Jankun, Ewa ewa.skrzypczak-jan...@utoledo.edu schrieb am Do, 28.10.2010: Von: Skrzypczak-Jankun, Ewa ewa.skrzypczak-jan...@utoledo.edu Betreff: Re: [ccp4bb] Additional band on gel due to his-tag: any references? An: CCP4BB@JISCMAIL.AC.UK Datum: Donnerstag, 28. Oktober, 2010 16:09 Uhr Additional band on a gel might not be caused by his-tag. It is often a result of different conformation/molecular shape and so the molecule travels with different speed in the gel. We may wish for a homogenous sample (chemically and structurally) but this is seldom true See example: Int J Mol Med 2009, 23(1), 57 Jankun et al, VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form. I fully agree with Tim - making rude comments about reviewers (or anybody else) is not going to help you. Questioning an extra band is a legitimate remark that you should address. Good luck - Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Thursday, October 28, 2010 7:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Additional band on gel due to his-tag: any references? Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since referees know the names of the authors of the articles they are refereeing, you may need a lot more than a couple of references to appease them after you called them bloody-minded in a public email forum. My two cents, Tim On Thu, Oct 28, 2010 at 01:16:59PM +0200, Sebastiaan Werten wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
It can sometimes be struggle to find the boundary between cynicism and pragmatism! I was, however, rather bemused by Dr Joosten's 7 rules of thumb - probably all of which I use and have seen used by referees. Of course I wouldn't want to blindly advocate any of them, however their use does make life somewhat easier for those of us who use crystallography to discover things about biology compared with their use by the (rather impressive!) members of this community who are involved in theoretical/methodological development. A time comes on my projects where you have to say two things - 1) my structure is telling me x and although I can spend the next six months performing minor tweaks these will not add (or subtract) from the conclusions I am interested in and 2)when I submit this to referees will they think my structure is appropriate to draw these conclusions?. It is whilst asking these two questions that rules of thumb become somewhat handy, especially when they coincide with the rules of thumb used by the referees. Simon On 28 Oct 2010, at 10:28, Eleanor Dodson wrote: Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the best model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little to no impact on the biology being described. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. Simon On 27 Oct 2010, at 20:11, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Young and Impressionable readers: I second-guess here that Robbie's intent - after re-refining many many PDB structures, seeing dreadful things, and becoming a hardened cynic - is to provoke more discussion in order to put in perspective - if not debunk- almost all of these rules. So it may be better to pretend you have never heard of these rules. Your crystallographic life might be a happier and less biased one. If you follow this simple procedure (not a rule) The model that fits the primary evidence (minimally biased electron density) best and is at the same time physically meaningful, is the best model, i. e., all plausibly accountable electron density (and not more) is modeled. This process of course does require a little work (like looking through all of the model, not just the interesting parts, and thinking what makes sense) but may lead to additional and unexpected insights. And in almost all cases, you will get a model with plausible statistics, without any reliance on rules. For some decisions regarding global parameterizations you have to apply more sophisticated test such as Ethan pointed out (HR tests) or Ian uses (LL-tests). And once you know how to do that, you do not need any rules of thumb anyhow. So I opt for a formal burial of these rules of thumb and a toast to evidence and plausibility. And, as Gerard B said in other words so nicely: Si tacuisses, philosophus mansisses. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robbie Joosten Sent: Tuesday, October 26, 2010 10:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Dear Anthony, That is an excellent question! I believe there are quite a lot of 'rules of thumb' going around. Some of them seem to lead to very dogmatic thinking and have caused (refereeing) trouble for good structures and lack of trouble for bad structures. A lot of them were discussed at the CCP4BB so it may be nice to try to list them all. Rule 1: If Rwork 20%, you are done. Rule 2: If R-free - Rwork 5%, your structure is wrong. Rule 3: At resolution X, the bond length rmsd should be than Y (What is the rmsd thing people keep talking about?) Rule 4: If your resolution is lower than X, you should not use_anisotropic_Bs/riding_hydrogens Rule 5: You should not build waters/alternates at resolutions lower than X Rule 6: You should do the final refinement with ALL reflections Rule 7: No one cares about getting the carbohydrates right Obviously, this list is not complete. I may also have overstated some of the rules to get the discussion going. Any addidtions are welcome. Cheers, Robbie Joosten Netherlands Cancer Institute Apologies if I have missed a recent relevant thread, but are lists of rules of thumb for model building and refinement? Anthony Anthony Duff Telephone: 02 9717 3493 Mob: 043 189 1076 =
Re: [ccp4bb] Bug in c_truncate?
Hello Peter, I faintly rememeber a similar kind of problem, and think that if you replace -1 with 0, the problem should go away. It seemed that -1 is not an allowed flag for (some) ccp4 programs. Please let us know if this resolves the issue. Tim On Thu, Oct 28, 2010 at 10:21:20AM -0400, Peter Chan wrote: Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried looking for known/resolved issues/updates in version 6.1.3 but could not find any so I assumed it is the same version of f2mtz/ctruncate/uniqueify. I used the GUI version of F2MTZ, with the settings below: - import file in SHELX format - keep existing FreeR flags - fortran format (3F4.0,2F8.3,F4.0) - added data label I other integer // FreeRflag The hkl file, in SHELX format, output by XPREP look something like this: -26 -3 1 777.48 39.19 26 -3 -1 800.83 36.31 -26 3 -1 782.67 37.97 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 Notice the test set is flagged -1 and the working set is not flagged at all. This actually lead to another error message in f2mtz about missing FreeR flags. From my understanding, the SHELX flagging convention is 1 for working and -1 for test. So I manually tagged the working set with 1 using vi: -26 -3 1 777.48 39.19 1 26 -3 -1 800.83 36.31 1 -26 3 -1 782.67 37.97 1 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 This is the file which gives me the error message: Problem with FREE column in input file. All flags apparently identical. Check input file.. Apparently, import to mtz works ok when I use old-truncate instead of c-truncate. Best, Peter -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Against Method (R)
In addition to bulk solvent, the other well recognized problem with macromolecular structures is the inadequate description of disorder. With small molecules, the Debye-Waller works much better because the harmonic oscillator is indeed a good model there. Note that the problem is not anisotropy (which we can model if resolution is sufficiently high), but rather anharmonic motion and multiple conformations that go undetected. On Thu, 2010-10-28 at 08:00 -0500, Jacob Keller wrote: So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:05 AM Subject: Re: [ccp4bb] Against Method (R) It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives similar results to R(sigma), both attempt to measure the precision of the MERGED data, which are what one is refining against. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 27 Oct 2010, Ed Pozharski wrote: On Tue, 2010-10-26 at 21:16 +0100, Frank von Delft wrote: the errors in our measurements apparently have no bearing whatsoever on the errors in our models This would mean there is no point trying to get better crystals, right? Or am I also wrong to assume that the dataset with higher I/sigma in the highest resolution shell will give me a better model? On a related point - why is Rmerge considered to be the limiting value for the R? Isn't Rmerge a poorly defined measure itself that deteriorates at least in some circumstances (e.g. increased redundancy)? Specifically, shouldn't ideal R approximate 0.5*sigmaI/I? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs *** 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 *** -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Against Method (R)
Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Thu, 28 Oct 2010, Jacob Keller wrote: So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:05 AM Subject: Re: [ccp4bb] Against Method (R) It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives similar results to R(sigma), both attempt to measure the precision of the MERGED data, which are what one is refining against. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 27 Oct 2010, Ed Pozharski wrote: On Tue, 2010-10-26 at 21:16 +0100, Frank von Delft wrote: the errors in our measurements apparently have no bearing whatsoever on the errors in our models This would mean there is no point trying to get better crystals, right? Or am I also wrong to assume that the dataset with higher I/sigma in the highest resolution shell will give me a better model? On a related point - why is Rmerge considered to be the limiting value for the R? Isn't Rmerge a poorly defined measure itself that deteriorates at least in some circumstances (e.g. increased redundancy)? Specifically, shouldn't ideal R approximate 0.5*sigmaI/I? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs *** 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] Additional band on gel due to his-tag: any references?
Hi Seb, I'm not aware of the notion (and neither are your reviewers apparently) that a His tag often results in two bands on a lane in SDS page. Why would that be? Extra SDS binding to the positive patch? Just wondering if there's any truth to your statement. Also, since in this case there seems to be consensus (!) among reviewers regarding the double band, they may have a legitimate point..my 2 cents. Regards, Bert On 10/28/10 7:16 AM, Sebastiaan Werten sebastiaan.wer...@uni-greifswald.de wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de
Re: [ccp4bb] Babinet solvent correction [WAS: R-free flag problem]
Hi Tim, sorry for my late reply - I just came back to the lab. In the Babinet bulk solvent correction, no bulk solvent phases are used, it is entirely based on amplitudes and strictly only valid if the phases of the bulk solvent are opposite to the ones of the protein. And as Sasha Urzhumtsev pointed out, this assumption is only valid at very low resolution. The mask bulk solvent correction is a vector sum including the phases of the bulk solvent mask, which makes a difference at medium resolution (up to ~4.5 A, or so). As far as I can see, your formulas given below do not distinguish between amplitude (modulus) and vector bulk solvent corrections. Personally, I really don't see any physical sense in using both corrections together, except for compensating any potential scaling problems at low resolution. If the model is basically complete and correct, the mask bulk solvent correction is usually superior to the Babinet bulk solvent correction (see, for example, my old and small CCP4 Newsletter contribution http://www.ccp4.ac.uk/newsletters/newsletter34/bsdk_text.html). However, there are also good reasons for using the Babinet bulk solvent correction (it should be an option in ALL refinement programs!): - it requires only two parameters and can be used in any case - in rigid body refinement, the mask lags behind; here, I always use the Babinet BS correction - channels could show false positive density, because the mask left them empty - this depends heavily on the choice of radii to determine/shrink the bulk solvent mask; in such cases, I always calculate a Babinet BS correction as a control Best regards, Dirk. Am 23.10.10 22:14, schrieb Tim Fenn: On Sat, 23 Oct 2010 10:05:15 -0700 Pavel Afoninepafon...@gmail.com wrote: Hi Tim, ...but I hope this answers the question: Babinet's vs. the flat model? Use them together! ;) thanks a lot for your reply. Could you please explain the *physical* meaning of using both models together? I can try! Typically, we model the bulk solvent using a real space mask that is set to 1 in the bulk solvent region and 0 in the protein. This gets Fourier transformed, symmetrized and added in to the scattering factors from the molecule (Equation 1 in the paper, page 6 in your presentation): Ftot = Fc + ks*Fs*exp(-Bs*s^2/4) which works great and is how things are usually coded in most macromolecular software, no problems or arguments there. However, we can come from the opposite - but equivalent! - direction of Babinet's principle, which tells us the bulk solvent can also be modeled by inverting everything: set the bulk solvent region to 0 and the protein region to 1 in the real space mask, apply a Fourier transform to that and then invert the phase: Ftot = Fc - ks*Fm*exp(-Bs*s^2/4) (I'm using Fm to distinguish it from Fs, due to the inversion of 0's and 1's in the real space mask) This is equation 2 in the paper. So we're still using the flat model to compute Fm, and we're using Babinet's principle to add it in to the structure factors - although its better described as adding the inverse (thus the minus sign in the second equation) of the complement (Fm rather than Fs). These two equations are exactly equivalent, without any loss of generality. So, I would argue the flat model and Babinet's are very much congruous. Also take a look at the description/discussion in the paper regarding Figure 2 (which helped me think about things at first). The big difference is that Babinet's is usually applied as: Ftot = Fc - ks*Fc*exp(-Bs*s^2/4) which, I would argue, isn't quite right - the bulk solvent doesn't scatter like protein, but it does get the shape right. Which I think is why Fokine and Urzhumtsev point out that at high resolution this form would start to show disagreement with the data. I haven't looked at this explicitly though, so we still haven't answered that question! We didn't want to spend much time on it in the paper, our main goal was to try out the differentiable models we describe. The Babinet trick was a convenient way to make coding easier. Anyway, I hope this helps explain it a bit more, and again: sorry for the long-windedness. Regards, Tim -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
Please let me clarify that it is by no means my intention to be rude to any referees, nor to round up alternative explanations for the extra band. The only thing I am after is a proper reference for the phenomenon of his-tagged proteins producing an extra band at slightly higher apparent molecular weight. I just unearthed a 2007 thread from the CCP4 archives where this very subject appears to have been discussed already (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0711L=CCP4BBD=0P=38996) but unfortunately I didn't find any references to published literature there either. Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de On Thu, 28 Oct 2010 10:09:37 -0400, Skrzypczak-Jankun, Ewa ewa.skrzypczak-jan...@utoledo.edu wrote: Additional band on a gel might not be caused by his-tag. It is often a result of different conformation/molecular shape and so the molecule travels with different speed in the gel. We may wish for a homogenous sample (chemically and structurally) but this is seldom true See example: Int J Mol Med 2009, 23(1), 57 Jankun et al, VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form. I fully agree with Tim - making rude comments about reviewers (or anybody else) is not going to help you. Questioning an extra band is a legitimate remark that you should address. Good luck - Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tim Gruene Sent: Thursday, October 28, 2010 7:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Additional band on gel due to his-tag: any references? Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since referees know the names of the authors of the articles they are refereeing, you may need a lot more than a couple of references to appease them after you called them bloody-minded in a public email forum. My two cents, Tim On Thu, Oct 28, 2010 at 01:16:59PM +0200, Sebastiaan Werten wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de --
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
Dear Sebastian, Having personally purified upwards of 500 (I lost count really) of His-tagged proteins, I can't say that I have the same awareness as you with respect to the additional band being 'very common'. Depending on the kind of expression system, size of your protein, conditions of purification, and most importantly the kind of IMAC resin you're using there can be anywhere between zero and five *extra* bands resulting from contaminants binding to the resin. In the case of E. coli expression there usually are two main contaminants (SlyD and a 61 kDa protein) but can be more than that if the ratio of your protein to resin is unfavorable. Without going through a huge PITA you can't be sure that the extra band is even related to your protein of interest - for example, you could do an in-gel digest and get peptides identified by MS however if your extra band is close enough to the main band there will always be a lingering concern that your main band is 'bleeding' into the extra band and therefore the in-gel digest results are biased towards the expected. What sort of a concern are these reviewers trying to address? Is there some unique biological activity in the sample (like a novel enzyme activity previously unobserved for your target protein) - in which case the concerns might be justified, or is it just the level of purity with respect to structure being solved - in which case it's not much of a concern at all? Artem On Thu, Oct 28, 2010 at 6:16 AM, Sebastiaan Werten sebastiaan.wer...@uni-greifswald.de wrote: Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the additional band is a very common artifact with his-tagged proteins, but I was wondering if anyone is aware of a paper that formally describes the phenomenon, as we need to appease a couple of rather bloody-minded referees. Thanks very much for any suggestions, Seb. -- Dr. Sebastiaan Werten Institut für Biochemie Universität Greifswald Felix-Hausdorff-Str. 4 D-17489 Greifswald Germany Tel: +49 38 34 86 44 61 E-mail: sebastiaan.wer...@uni-greifswald.de
Re: [ccp4bb] Additional band on gel due to his-tag: any references?
THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN June 14, 2002 The Journal of Biological Chemistry, 277, 21397-21404. His-tagged Iron cluster that runs as a doublet. Mass-spec show they are the same species. They concluded that the protein binds SDS in two stoichiometries and therefore runs as a doublet as seen for the OmpA protein (references are in the paper). Hope that helps. Dan
Re: [ccp4bb] Against Method (R)
So I guess there is never a case in crystallography in which our models predict the data to within the errors of data collection? I guess the situation might be similar to fitting a Michaelis-Menten curve, in which the fitted line often misses the error bars of the individual points, but gets the overall pattern right. In that case, though, I don't think we say that we are inadequately modelling the data. I guess there the error bars are actually too small (are underestimated.) Maybe our intensity errors are also underestimated? JPK On Thu, Oct 28, 2010 at 9:50 AM, George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de wrote: Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Thu, 28 Oct 2010, Jacob Keller wrote: So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:05 AM Subject: Re: [ccp4bb] Against Method (R) It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives similar results to R(sigma), both attempt to measure the precision of the MERGED data, which are what one is refining against. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 27 Oct 2010, Ed Pozharski wrote: On Tue, 2010-10-26 at 21:16 +0100, Frank von Delft wrote: the errors in our measurements apparently have no bearing whatsoever on the errors in our models This would mean there is no point trying to get better crystals, right? Or am I also wrong to assume that the dataset with higher I/sigma in the highest resolution shell will give me a better model? On a related point - why is Rmerge considered to be the limiting value for the R? Isn't Rmerge a poorly defined measure itself that deteriorates at least in some circumstances (e.g. increased redundancy)? Specifically, shouldn't ideal R approximate 0.5*sigmaI/I? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs *** 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] Bug in c_truncate?
Hello Tim, Thank you for the suggestion. I have now tagged the working set as 1 and test set as 0. Unfortunately, it still gives the same error about all Rfree being the same, and only in c-truncate but not old-truncate. Perhaps I should install 6.1.3 and see if the problem still persist. Best, Peter Date: Thu, 28 Oct 2010 16:29:31 +0200 From: t...@shelx.uni-ac.gwdg.de Subject: Re: [ccp4bb] Bug in c_truncate? To: CCP4BB@JISCMAIL.AC.UK Hello Peter, I faintly rememeber a similar kind of problem, and think that if you replace -1 with 0, the problem should go away. It seemed that -1 is not an allowed flag for (some) ccp4 programs. Please let us know if this resolves the issue. Tim On Thu, Oct 28, 2010 at 10:21:20AM -0400, Peter Chan wrote: Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried looking for known/resolved issues/updates in version 6.1.3 but could not find any so I assumed it is the same version of f2mtz/ctruncate/uniqueify. I used the GUI version of F2MTZ, with the settings below: - import file in SHELX format - keep existing FreeR flags - fortran format (3F4.0,2F8.3,F4.0) - added data label I other integer // FreeRflag The hkl file, in SHELX format, output by XPREP look something like this: -26 -3 1 777.48 39.19 26 -3 -1 800.83 36.31 -26 3 -1 782.67 37.97 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 Notice the test set is flagged -1 and the working set is not flagged at all. This actually lead to another error message in f2mtz about missing FreeR flags. From my understanding, the SHELX flagging convention is 1 for working and -1 for test. So I manually tagged the working set with 1 using vi: -26 -3 1 777.48 39.19 1 26 -3 -1 800.83 36.31 1 -26 3 -1 782.67 37.97 1 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 This is the file which gives me the error message: Problem with FREE column in input file. All flags apparently identical. Check input file.. Apparently, import to mtz works ok when I use old-truncate instead of c-truncate. Best, Peter -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] Bug in c_truncate?
Why are you running [c]truncate? this is used to convert I - F and I would be surprised if it recognised or preserved a FreeR column Phil On 28 Oct 2010, at 17:48, Peter Chan wrote: Hello Tim, Thank you for the suggestion. I have now tagged the working set as 1 and test set as 0. Unfortunately, it still gives the same error about all Rfree being the same, and only in c-truncate but not old-truncate. Perhaps I should install 6.1.3 and see if the problem still persist. Best, Peter Date: Thu, 28 Oct 2010 16:29:31 +0200 From: t...@shelx.uni-ac.gwdg.de Subject: Re: [ccp4bb] Bug in c_truncate? To: CCP4BB@JISCMAIL.AC.UK Hello Peter, I faintly rememeber a similar kind of problem, and think that if you replace -1 with 0, the problem should go away. It seemed that -1 is not an allowed flag for (some) ccp4 programs. Please let us know if this resolves the issue. Tim On Thu, Oct 28, 2010 at 10:21:20AM -0400, Peter Chan wrote: Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried looking for known/resolved issues/updates in version 6.1.3 but could not find any so I assumed it is the same version of f2mtz/ctruncate/uniqueify. I used the GUI version of F2MTZ, with the settings below: - import file in SHELX format - keep existing FreeR flags - fortran format (3F4.0,2F8.3,F4.0) - added data label I other integer // FreeRflag The hkl file, in SHELX format, output by XPREP look something like this: -26 -3 1 777.48 39.19 26 -3 -1 800.83 36.31 -26 3 -1 782.67 37.97 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 Notice the test set is flagged -1 and the working set is not flagged at all. This actually lead to another error message in f2mtz about missing FreeR flags. From my understanding, the SHELX flagging convention is 1 for working and -1 for test. So I manually tagged the working set with 1 using vi: -26 -3 1 777.48 39.19 1 26 -3 -1 800.83 36.31 1 -26 3 -1 782.67 37.97 1 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 This is the file which gives me the error message: Problem with FREE column in input file. All flags apparently identical. Check input file.. Apparently, import to mtz works ok when I use old-truncate instead of c-truncate. Best, Peter -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
2) when I submit this to referees will they think my structure is appropriate to draw these conclusions?. Particularly question 2) can rarely be answered w/o looking at electron density. Binding site details, ligand geometry, all have practically no effect on the Rules of Thumb. It is whilst asking these two questions that rules of thumb become somewhat handy, especially when they coincide with the rules of thumb used by the referees. Keep also in mind that every atom contributes to every reflection. So fixing ANY error WHEREVER will improve you refinement - also in the part you are actually interested in. You point is correct however that once your question is answered you could stop. That however does not necessarily deliver a publishable structure that might be used by others for purposes you initially did not intend. Pharma industry does this all the time quickly looking for ligands and abandoning pointless structures early - but they don't publish the unfinished models. br On 28 Oct 2010, at 10:28, Eleanor Dodson wrote: Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the best model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little to no impact on the biology being described. Mind you discussions on this email list can be a useful resource for telling referee's why you don't think you should comply with their rule of thumb. Simon On 27 Oct 2010, at 20:11, Bernhard Rupp (Hofkristallrat a.D.) wrote: Dear Young and Impressionable readers: I second-guess here that Robbie's intent - after re-refining many many PDB structures, seeing dreadful things, and becoming a hardened cynic - is to provoke more discussion in order to put in perspective - if not debunk- almost all of these rules. So it may be better to pretend you have never heard of these rules. Your crystallographic life might be a happier and less biased one. If you follow this simple procedure (not a rule) The model that fits the primary evidence (minimally biased electron density) best and is at the same time physically meaningful, is the best model, i. e., all plausibly accountable electron density (and not more) is modeled. This process of course does require a little work (like looking through all of the model, not just the interesting parts, and thinking what makes sense) but may lead to additional and unexpected insights. And in almost all cases, you will get a model with plausible statistics, without any reliance on rules. For some decisions regarding global parameterizations you have to apply more sophisticated test such as Ethan pointed out (HR tests) or Ian uses (LL-tests). And once you know how to do that, you do not need any rules of thumb anyhow. So I opt for a formal burial of these rules of thumb and a toast to evidence and plausibility. And, as Gerard B said in other words so nicely: Si tacuisses, philosophus mansisses. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robbie Joosten Sent: Tuesday, October 26, 2010 10:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Dear Anthony, That is an excellent question! I believe there are quite a lot of 'rules of thumb' going around. Some of them seem to lead to very dogmatic thinking and have caused (refereeing) trouble for good structures and lack of trouble for bad structures. A lot of them were discussed at the CCP4BB so it may be nice to try to list them all. Rule 1: If Rwork 20%, you are done. Rule 2: If R-free - Rwork 5%, your structure is wrong. Rule 3: At resolution X, the bond length rmsd should be than Y (What is the rmsd thing people keep talking about?) Rule 4: If your resolution is lower than X, you should not use_anisotropic_Bs/riding_hydrogens Rule 5: You should not build waters/alternates at resolutions lower than X Rule 6: You should do the final refinement with ALL reflections Rule 7: No one cares about getting the carbohydrates right Obviously, this list is not complete. I may also have overstated some of the rules to get the discussion going. Any addidtions are welcome. Cheers, Robbie Joosten Netherlands Cancer Institute Apologies if I have missed a recent relevant thread, but are lists of rules of thumb for model building and refinement? Anthony Anthony Duff Telephone: 02 9717 3493 Mob: 043 189 1076 =
Re: [ccp4bb] Against Method (R)
It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where sigma is the rms error), then you expect the right curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be kicked further than 1 away from the right curve. In this case, a data point more than 1 away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong error model). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the R factor gap: solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically important systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to correct, what I would like to find is a structure that represents the transition between the small molecule and the macromolecule world. Lysozyme does not qualify! Even the famous 0.6 A structure of lysozyme (2vb1) still has a mean absolute chi: |Iobs-Icalc|/sig(I) = 4.5. Also, the 1.4 A structure of the tetrapeptide QQNN (2olx) is only a little better at |chi| = 3.5. I realize that the chi I describe here is not a standard crystallographic statistic, and perhaps I need a statistics lesson, but it seems to me there ought to be a case where it is close to 1. -James Holton MAD Scientist On Thu, Oct 28, 2010 at 9:04 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: So I guess there is never a case in crystallography in which our models predict the data to within the errors of data collection? I guess the situation might be similar to fitting a Michaelis-Menten curve, in which the fitted line often misses the error bars of the individual points, but gets the overall pattern right. In that case, though, I don't think we say that we are inadequately modelling the data. I guess there the error bars are actually too small (are underestimated.) Maybe our intensity errors are also underestimated? JPK On Thu, Oct 28, 2010 at 9:50 AM, George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de wrote: Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Thu, 28 Oct 2010, Jacob Keller wrote: So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:05 AM Subject: Re: [ccp4bb] Against Method (R) It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and sigmaI/I, which it calls R(sigma). For well-behaved crystals R1 is in the range 1-5% and R(merge) (based on intensities) is in the range 3-9%. As you suggest, 0.5*R(sigma) could be regarded as the lower attainable limit for R1 and this is indeed the case in practice (the factor 0.5 approximately converts from I to F). Rpim gives
Re: [ccp4bb] Bug in c_truncate?
The GUI task has the option to run (c)truncate after f2mtz (if you have intensities in the input hkl file), and then uniqueify after that. I can reproduce this problem. ctruncate is losing the freeR column. At the moment, I don't know if this is a bug or a feature. As a work around, you can run ctruncate for the analyses, and re-run with truncate for the MTZ file. Tim is right, you need to use 0 instead of -1 in the CCP4 convention. HTH Martyn PS Refmac is moving towards using intensities, so that you can avoid this step. But I believe 5.5 only uses intensities for twin refinement. On Thu, 2010-10-28 at 17:55 +0100, Phil Evans wrote: Why are you running [c]truncate? this is used to convert I - F and I would be surprised if it recognised or preserved a FreeR column Phil On 28 Oct 2010, at 17:48, Peter Chan wrote: Hello Tim, Thank you for the suggestion. I have now tagged the working set as 1 and test set as 0. Unfortunately, it still gives the same error about all Rfree being the same, and only in c-truncate but not old-truncate. Perhaps I should install 6.1.3 and see if the problem still persist. Best, Peter Date: Thu, 28 Oct 2010 16:29:31 +0200 From: t...@shelx.uni-ac.gwdg.de Subject: Re: [ccp4bb] Bug in c_truncate? To: CCP4BB@JISCMAIL.AC.UK Hello Peter, I faintly rememeber a similar kind of problem, and think that if you replace -1 with 0, the problem should go away. It seemed that -1 is not an allowed flag for (some) ccp4 programs. Please let us know if this resolves the issue. Tim On Thu, Oct 28, 2010 at 10:21:20AM -0400, Peter Chan wrote: Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried looking for known/resolved issues/updates in version 6.1.3 but could not find any so I assumed it is the same version of f2mtz/ctruncate/uniqueify. I used the GUI version of F2MTZ, with the settings below: - import file in SHELX format - keep existing FreeR flags - fortran format (3F4.0,2F8.3,F4.0) - added data label I other integer // FreeRflag The hkl file, in SHELX format, output by XPREP look something like this: -26 -3 1 777.48 39.19 26 -3 -1 800.83 36.31 -26 3 -1 782.67 37.97 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 Notice the test set is flagged -1 and the working set is not flagged at all. This actually lead to another error message in f2mtz about missing FreeR flags. From my understanding, the SHELX flagging convention is 1 for working and -1 for test. So I manually tagged the working set with 1 using vi: -26 -3 1 777.48 39.19 1 26 -3 -1 800.83 36.31 1 -26 3 -1 782.67 37.97 1 27 -3 1 45.722 25.711 -1 -27 3 1 -14.20 31.69 -1 This is the file which gives me the error message: Problem with FREE column in input file. All flags apparently identical. Check input file.. Apparently, import to mtz works ok when I use old-truncate instead of c-truncate. Best, Peter -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603634Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
[ccp4bb] Free R with doubled cell edge
Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two-fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a=30 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high temp simulated annealing and hope that any bias is gone. I'm not sure that I am comfortable with the word hope here... But, if the consensus of opinion of the wise folk at the BB is that this will pass muster at the point where the charming and delightful referees are commenting on the extremely high impact (obviously :-) manuscript, then I will quote you all! I await your wise words. Free R. Again. Sorry. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- A new scientific truth does not triumph by convincing opponents and making them see the light, but rather because its opponents eventually die, and a new generation grows up that is familiar with it. ~Max Planck
[ccp4bb] oligomer ligand building
Hi, all I am trying to build an oligomer ligand. I have obtained all the cif files for the monomers from HIC-UP. I tried to build several monomers in using coot. Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC 1 Y ABC 2X-Y). However, when using refmac to refine, it always separated the monomers and broke the bond, which I could fix it back in coot using 'real space refine zone'. I am wondering, what is the general practice to build such oligomer ligand? All suggestions are welcome. thank you! Changyi
Re: [ccp4bb] oligomer ligand building
Hi Changyi, I'm not sure if this will answer your question, but it is likely that the cif file you are reading into Refmac is not in the correct format from Hic-Cup. I would suggest reading your coordinate file for the ligand into the Dundee server which will output a cif formats for refinement programs. Scroll down to the Refmac output and save as a .cif, and then read this file into your next round of refinement with Refmac. After this round, Refmac will generate a new cif library containing this ligand. I hope this helps. Cheers, Paul
Re: [ccp4bb] Help with Optimizing Crystals
Hi. Thanks for all of the helpful advice. Below is a summary of the suggestions, along with some things that I have tried and the results thus far. 1) Make sure that the crystals are protein and not salt. My crystals absorb Izit dye well and shooting some initial crystals did not produce any diffraction. I have yet to see them on a gel though, possibly due to too few crystals run, loss of crystals while washing, etc. 2) Try streak seeding using the hit conditions as well as new conditions. Streak seeding using two hit conditions and half of the protein concentration have thus far only produced crystals that are smaller than the initial crystals used to make the seed stock. This indicates that self nucleation is still occurs. I will try significantly lowering the precipitant conc. as well. Seeding into a few random conditions has produced some crystals in new conditions, but they look the same or worse that the previous ones. 3) Make sure that the protein is very pure. Include an additional purification step to your purification scheme, try thermal denaturation of contaminating proteins, crystallize out protein and the redissolve the crystals and use to set up new trays. I will try some of these. 4) Modify the protein to make it more amendable to crystallization. Try truncations, mutations, methylate lysines. I tried the FL version from two different organisms and neither crystallizes. When enzymatically proteolyzed, the protein from one organism crystallizes. However, when proteins containing similar truncations are purified recombinately, a ladder of bands appears after the major one, indicating protein instability. I have not tried any mutations or methylation yet. Thanks for all of the helpful suggestions, and if there are any more, please let me know. Matt On Wed, Oct 27, 2010 at 9:28 AM, Annie Hassell annie.m.hass...@gsk.comwrote: Matt— You might want to try heating your protein to get rid of unfolded/improperly folded protein. We have used 37C for 10 min with good success, but a time course at different temperatures is the best way to determine which parameters are optimum for your protein. Heat—chill it on ice—centrifuge-- then set up your crystallization trays. It’s a pretty quick test to see if this will work for your protein. Do you have any ligands for your protein? These have often been the key to getting good crystals in our lab. If you do have good ligands, you may want to express and/or purify your protein in the presence of these compounds. Good Luck! annie *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Jürgen Bosch *Sent:* Tuesday, October 26, 2010 5:46 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Help with Optimizing Crystals Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. Resource Q ? two or more species perhaps ? Does it run as a monomer dimer multimer on your SEC ? 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. Do they float or do they sink quickly when you try to mount them ? 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. The additive screen from Hampton is not bad and can make a big difference. A different topic is it a direct cryo what you are using as a condition ? If not what do you use a s a cryo ? Have you tried the old-fashioned way of shooting at crystals at room temperature using capillaries (WTHIT ?) You might be killing your crystal by trying to cryo it is what I'm trying to say here. Jürgen Thanks for all the helpful advice thus far, Matt
Re: [ccp4bb] Babinet solvent correction [WAS: R-free flag problem]
On Thu, 28 Oct 2010 16:56:42 +0200 Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: In the Babinet bulk solvent correction, no bulk solvent phases are used, it is entirely based on amplitudes and strictly only valid if the phases of the bulk solvent are opposite to the ones of the protein. And as Sasha Urzhumtsev pointed out, this assumption is only valid at very low resolution. The mask bulk solvent correction is a vector sum including the phases of the bulk solvent mask, which makes a difference at medium resolution (up to ~4.5 A, or so). As far as I can see, your formulas given below do not distinguish between amplitude (modulus) and vector bulk solvent corrections. Sorry - I didn't make that clear. The formulas all use complex structure factors, as in the paper. Personally, I really don't see any physical sense in using both corrections together, except for compensating any potential scaling problems at low resolution. We're not using both corrections together - the Babinet *method* is used to add in the bulk solvent contribution computed using the flat mask *model* (or the polynomial/Gaussian model in the paper). The protein structure factors (Fc) are not used in the bulk solvent correction - nor, in my opinion, should they be (as I attempted to point out in my previous email). Regards, Tim -- - Tim Fenn f...@stanford.edu Stanford University, School of Medicine James H. Clark Center 318 Campus Drive, Room E300 Stanford, CA 94305-5432 Phone: (650) 736-1714 FAX: (650) 736-1961 -
Re: [ccp4bb] Against Method (R)
There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the "R factor gap" is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Bart On 10-10-28 11:33 AM, James Holton wrote: It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where "sigma" is the rms error), then you expect the "right" curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be "kicked" further than "1" away from the "right" curve. In this case, a data point more than "1" away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong "error model"). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the "R factor gap": solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically "important" systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to "correct", what I would like to find is a structure that represents the transition between the "small molecule" and the "macromolecule" world. Lysozyme does not qualify! Even the famous 0.6 A structure of lysozyme (2vb1) still has a "mean absolute chi": |Iobs-Icalc|/sig(I) = 4.5. Also, the 1.4 A structure of the tetrapeptide QQNN (2olx) is only a little better at |chi| = 3.5. I realize that the "chi" I describe here is not a "standard" crystallographic statistic, and perhaps I need a statistics lesson, but it seems to me there ought to be a case where it is close to 1. -James Holton MAD Scientist On Thu, Oct 28, 2010 at 9:04 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: So I guess there is never a case in crystallography in which our models predict the data to within the errors of data collection? I guess the situation might be similar to fitting a Michaelis-Menten curve, in which the fitted line often misses the error bars of the individual points, but gets the overall pattern right. In that case, though, I don't think we say that we are inadequately modelling the data. I guess there the error bars are actually too small (are underestimated.) Maybe our intensity errors are also underestimated? JPK On Thu, Oct 28, 2010 at 9:50 AM, George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de wrote: Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Thu, 28 Oct 2010, Jacob Keller wrote: So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/additionally, I wonder whether there also might be more variability molecule-to-molecule in proteins, which we may not model well either. JPK - Original Message - From: "George M. Sheldrick" gshe...@shelx.uni-ac.gwdg.de To:
Re: [ccp4bb] oligomer ligand building
On 28/10/10 20:12, Changyi Xue wrote: Hi, all I am trying to build an oligomer ligand. I have obtained all the cif files for the monomers from HIC-UP. I tried to build several monomers in using coot. Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC 1 Y ABC 2X-Y). However, when using refmac to refine, it always separated the monomers and broke the bond, which I could fix it back in coot using 'real space refine zone'. I am wondering, what is the general practice to build such oligomer ligand? Having a LINK in the PDB will show the link in the graphics. Having a link description in the cif file will allow you to refine it (presuming that such a link is not already in the dictionary). This applies to any refinement program. If you want to refine the link with Coot, you will need to use Sphere Refinement. (IMHO) cif link descriptions are not straightforward (not for the first few times that you make one, that is). Paul.
Re: [ccp4bb] Against Method (R)
So let's say I take a 0.6 Ang structure, artificially introduce noise into corresponding Fobs to make the resolution go down to 2 Ang, and refine using the 0.6 Ang model--do I actually get R's better than the artificially-inflated sigmas? Or let's say I experimentally decrease I/sigma by attenuating the beam and collect another data set--same situation? JPK - Original Message - From: Bart Hazes To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:13 PM Subject: Re: [ccp4bb] Against Method (R) There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the R factor gap is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Bart On 10-10-28 11:33 AM, James Holton wrote: It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where sigma is the rms error), then you expect the right curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be kicked further than 1 away from the right curve. In this case, a data point more than 1 away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong error model). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the R factor gap: solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically important systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to correct, what I would like to find is a structure that represents the transition between the small molecule and the macromolecule world. Lysozyme does not qualify! Even the famous 0.6 A structure of lysozyme (2vb1) still has a mean absolute chi: |Iobs-Icalc|/sig(I) = 4.5. Also, the 1.4 A structure of the tetrapeptide QQNN (2olx) is only a little better at |chi| = 3.5. I realize that the chi I describe here is not a standard crystallographic statistic, and perhaps I need a statistics lesson, but it seems to me there ought to be a case where it is close to 1. -James Holton MAD Scientist On Thu, Oct 28, 2010 at 9:04 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: So I guess there is never a case in crystallography in which our models predict the data to within the errors of data collection? I guess the situation might be similar to fitting a Michaelis-Menten curve, in which the fitted line often misses the error bars of the individual points, but gets the overall pattern right. In that case, though, I don't think we say that we are inadequately modelling the data. I guess there the error bars are actually too small (are underestimated.) Maybe our intensity errors are also underestimated? JPK On Thu, Oct 28, 2010 at 9:50 AM, George M. Sheldrick gshe...@shelx.uni-ac.gwdg.de wrote: Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*sigmaI/I = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen,
Re: [ccp4bb] Against Method (R)
Yes, but even the high-resolution structures cannot explain THEIR data to within experimental error. You can see this if you download the CIF file for one of the highest-resolution structures there is: 2vb1 (triclinic lysozyme at 0.6 A), which contains both I and FC: http://www.rcsb.org/pdb/download/downloadFile.do?fileFormat=STRUCTFACTcompression=NOstructureId=2VB1 I even had Z. Dauter send me the original image files for this one, and I don't think it will surprise anyone to hear that I think he did it right. Nevertheless, the average value of |Iobs-Icalc| / sigma(Iobs) is 4.5 for this structure. Also, if I take data from MLFSOM-simulated diffraction images (including anomalous scattering, absorption, shutter jitter, background, etc.) and set ARP/wARP to work building the model, starting from the SAD-phased map, it inevitably converges to R/Rfree of around 6-7%, even at 2 A resolution. For the real data, however, it never gets below 18%. This is actually not all that remarkable a result, because the Fobs from the fake data is actually only ~5% different from Fcalc from the PDB file I put into the simulation. (I did not provide this PDB to ARP/wARP!) Add this to the fact that if your model is close, but missing a few bits, then those missing bits light up in a Fo-Fc map (like the tail on Kevin Cowtan's cat). These differnece features get BETTER as the model becomes more complete, and in small molecule structures adding in difference features eventually leads to R1 R(merge) (using Sheldrick's notation from below.) What I don't understand is why protein structures don't converge like this. Yes, there are low-occupancy features: Fraser et al. 2009: http://dx.doi.org/10.1038/nature08615 Lang et al. 2010: http://dx.doi.org/10.1002/pro.423 but even if you model these in, the R factor only drops a few percent: van den Bedem et al. 2009: http://dx.doi.org/10.110/S0907444909030613 -James Holton MAD Scientist On Thu, Oct 28, 2010 at 2:13 PM, Bart Hazes bart.ha...@ualberta.ca wrote: There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the R factor gap is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Bart On 10-10-28 11:33 AM, James Holton wrote: It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where sigma is the rms error), then you expect the right curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be kicked further than 1 away from the right curve. In this case, a data point more than 1 away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong error model). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the R factor gap: solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically important systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to correct, what I would like to find is a structure that represents the transition between the small molecule and the macromolecule world. Lysozyme does not qualify! Even the famous 0.6 A structure of lysozyme (2vb1) still has a mean absolute chi: |Iobs-Icalc|/sig(I) = 4.5. Also, the 1.4 A structure of the tetrapeptide QQNN (2olx) is only a little
Re: [ccp4bb] Against Method (R)
Bart Hazes wrote There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the R factor gap is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Excellent point. On Thursday, October 28, 2010 02:49:11 pm Jacob Keller wrote: So let's say I take a 0.6 Ang structure, artificially introduce noise into corresponding Fobs to make the resolution go down to 2 Ang, and refine using the 0.6 Ang model--do I actually get R's better than the artificially-inflated sigmas? Or let's say I experimentally decrease I/sigma by attenuating the beam and collect another data set--same situation? This I can answer based on experience. One can take the coordinates from a structure refined at near atomic resolution (~1.0A), including multiple conformations, partial occupancy waters, etc, and use it to calculate R factors against a lower resolution (say 2.5A) data set collected from an isomorphous crystal. The R factors from this total-rigid-body replacement will be better than anything you could get from refinement against the lower resolution data. In fact, refinement from this starting point will just make the R factors worse. What this tells us is that the crystallographic residuals can recognize a better model when they see one. But our refinement programs are not good enough to produce such a better model in the first place. Worsr, they are not even good enough to avoid degrading the model. That's essentially the same thing Bart said, perhaps a little more pessimistic :-) cheers, Ethan JPK - Original Message - From: Bart Hazes To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:13 PM Subject: Re: [ccp4bb] Against Method (R) There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the R factor gap is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Bart On 10-10-28 11:33 AM, James Holton wrote: It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where sigma is the rms error), then you expect the right curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be kicked further than 1 away from the right curve. In this case, a data point more than 1 away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong error model). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the R factor gap: solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically important systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to correct, what I would
Re: [ccp4bb] oligomer ligand building
Hi As I see you want to use link between ligands. You need to create this link description first. It can be done using JLigand that is available from: www.ysbl.york.ac.uk/mxstat/ There are tutorials how to create ligands and links. It should help you to create links We are updating at the moment JLigand and it would be good if you take it after this sunday when we will have newer version of JLigand. regards Garib On 28 Oct 2010, at 20:12, Changyi Xue wrote: Hi, all I am trying to build an oligomer ligand. I have obtained all the cif files for the monomers from HIC-UP. I tried to build several monomers in using coot. Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC 1 Y ABC 2X-Y). However, when using refmac to refine, it always separated the monomers and broke the bond, which I could fix it back in coot using 'real space refine zone'. I am wondering, what is the general practice to build such oligomer ligand? All suggestions are welcome. thank you! Changyi
Re: [ccp4bb] Against Method (R)
You're second suggestion would be a good test because you are dealing with data from the same crystal and can thus assume the structures are identical (radiation damage excluded). So, take a highly diffracting crystal and collect a short-exposure low resolution data set and long exposure high resolution data set. Let's say with I/Sig=2 at 2.0 and 1.2 high-resolution shells. Give the data to two equally capable students to determine the structure by molecular replacement from a, let's say 30% sequence identity starting model. You could also use automated model building to be more objective and avoid becoming unpopular with your students. Proceed until each model is fully refined against its own data. Now run some more refinement, without manual rebuilding, of the lowres model versus the highres data (and perhaps some rigid body or other minimal refinement of the highres model versus the lowres data, make sure R Rfree go down). I predict the highres model will fit the lowres data noticeably better than the lowres model did and the lowres model, even after refinement with the highres data, will not reach the same quality as the highres model. Looking at Fo-Fc maps in the latter case may give some hints as to which model errors were not recognized at 2A resolution. You'll probably find peptide flips, mis-modeled leucine and other side chains, dual conformations not recognized at 2A resolution, more realistic B values, more waters ... Bart On 10-10-28 03:49 PM, Jacob Keller wrote: So let's say I take a 0.6 Ang structure, artificially introduce noise into corresponding Fobs to make the resolution go down to 2 Ang, and refine using the 0.6 Ang model--do I actually get R's better than the artificially-inflated sigmas? Or let's say I experimentally decrease I/sigma by attenuating the beam and collect another data set--same situation? JPK - Original Message - From: Bart Hazes To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 28, 2010 4:13 PM Subject: Re: [ccp4bb] Against Method (R) There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in the "R factor gap" is many small errors that are introduced during model building and not recognized and fixed later due to limited resolution. In a perfect world, refinement would find the global minimum but in practice all these little errors get stuck in local minima with distortions in neighboring atoms compensating for the initial error and thereby hiding their existence. Bart On 10-10-28 11:33 AM, James Holton wrote: It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where "sigma" is the rms error), then you expect the "right" curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you expect a certain small number of the errors to be large. If the curve passes within the bounds of every single one of your error bars, then your error estimates are either too big, or the errors have a non-Gaussian distribution. For example, if the noise in the data somehow had a uniform distribution (always between +1 and -1), then no data point will ever be "kicked" further than "1" away from the "right" curve. In this case, a data point more than "1" away from the curve is evidence that you either have the wrong model (curve), or there is some other kind of noise around (wrong "error model"). As someone who has spent a lot of time looking into how we measure intensities, I think I can say with some considerable amount of confidence that we are doing a pretty good job of estimating the errors. At least, they are certainly not off by an average of 40% (20% in F). You could do better than that estimating the intensities by eye! Everybody seems to have their own favorite explanation for what I call the "R factor gap": solvent, multi-confomer structures, absorption effects, etc. However, if you go through the literature (old and new) you will find countless attempts to include more sophisticated versions of each of these hypothetically "important" systematic errors, and in none of these cases has anyone ever presented a physically reasonable model that explained the observed spot intensities from a protein crystal to within experimental error. Or at least, if there is such a paper, I haven't seen it. Since there are so many possible things to "correct", what I would like to find is a structure that represents the transition between the "small molecule" and the "macromolecule" world. Lysozyme does not qualify! Even the famous 0.6 A structure of lysozyme (2vb1) still has a "mean absolute chi": |Iobs-Icalc|/sig(I) = 4.5.
Re: [ccp4bb] Against Method (R)
On 10-10-28 04:09 PM, Ethan Merritt wrote: This I can answer based on experience. One can take the coordinates from a structure refined at near atomic resolution (~1.0A), including multiple conformations, partial occupancy waters, etc, and use it to calculate R factors against a lower resolution (say 2.5A) data set collected from an isomorphous crystal. The R factors from this total-rigid-body replacement will be better than anything you could get from refinement against the lower resolution data. In fact, refinement from this starting point will just make the R factors worse. What this tells us is that the crystallographic residuals can recognize a better model when they see one. But our refinement programs are not good enough to produce such a better model in the first place. Worsr, they are not even good enough to avoid degrading the model. That's essentially the same thing Bart said, perhaps a little more pessimistic :-) cheers, Ethan Not pessimistic at all, just realistic and perhaps even optimistic for methods developers as apparently there is still quite a bit of progress that can be made by improving the search strategy during refinement. During manual refinement I normally tell students not to bother about translating/rotating/torsioning atoms by just a tiny bit to make it fit better. Likewise there is no point in moving atoms a little bit to correct a distorted bond or bond length. If it needed to move that little bit the refinement program would have done it for you. Look for discreet errors in the problematic residue or its neighbors: peptide flips, 120 degree changes in side chain dihedrals, etc. If you can find and fix one of those errors a lot of the stereochemical distortions and non-ideal fit to density surrounding that residue will suddenly disappear as well. The benefit of high resolution is that it is much easier to pick up and fix such errors (or not make them in the first place) Bart -- Bart Hazes (Associate Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
Zbyszek, Since you mention I/sigmaI in your PDF, do you mean I/sigmaI or I/sigmaI? Do you mean I/sigmaI (in whatever rendition you choose) for the averaged unique reflections or the I/sigmaI for the observations? Also since one can adjust sigmaI in your scalepack program through the use of the Error Scale Factor or the Error Model, how can a reviewer believe any of the I/sigmaI that are reported by authors? Thanks for any insights into these questions, Jim -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Zbyszek Otwinowski Sent: Thursday, October 28, 2010 7:41 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Feel free to use it as you wish. -- DUFF, Anthony wrote: I reckon you could share hypothetical review comments for educational purposes. -Original Message- From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thu 10/28/2010 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Why not double open review? If I have something reasonable to say, I should be able to sign it. Particularly if the publicly purported point of review is to make the manuscript better. And imagine what wonderful open hostility we would enjoy instead of all these hidden grudges! You would never have to preemptively condemn a paper on grounds of suspicion that it is from someone who might have reviewed you equally loathful earlier. You actually know that you are creaming the right bastard! A more serious question for the editors amongst us: Can I publish review comments or are they covered under some confidentiality rule? Some of these gems are quite worthy public entertainment. Best, BR -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
Lake Wobegon!!! For those outside the US and/or otherwise not familiar with that small town, check out: http://en.wikipedia.org/wiki/Lake_Wobegon Lake Wobegon, where all the women are strong, all the men are good looking, and all the children are above average The best use of modern statistical concepts in a rebuttal (or in any paper, for that matter) I have seen in a long time! I totally support starting a collection of 'hilarious' reviewers' comments and rebuttals. Our resident KuK Hofkristallograf is probably correct in trying to establish first whether such Schmaeh is legal. If it is, let the flood gates burst! MM On Oct 28, 2010, at 8:40 PM, Zbyszek Otwinowski wrote: Feel free to use it as you wish. -- DUFF, Anthony wrote: I reckon you could share hypothetical review comments for educational purposes. -Original Message- From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thu 10/28/2010 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Why not double open review? If I have something reasonable to say, I should be able to sign it. Particularly if the publicly purported point of review is to make the manuscript better. And imagine what wonderful open hostility we would enjoy instead of all these hidden grudges! You would never have to preemptively condemn a paper on grounds of suspicion that it is from someone who might have reviewed you equally loathful earlier. You actually know that you are creaming the right bastard! A more serious question for the editors amongst us: Can I publish review comments or are they covered under some confidentiality rule? Some of these gems are quite worthy public entertainment. Best, BR -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu REVIEW_CRITERIA.pdf
[ccp4bb] Post-Doctoral Position in Structural Biology
Dear Friends: One post-doctoral http://www.riken.go.jp/engn/r-world/info/recruit/k101029_s_rsc.html position to work on structural biology project is available immediately at RIKEN SPring-8 Center, Harima Institute, Japan. With regards, Kumarevel ** Dr Thirumananseri KUMAREVEL Senior Research Scientist Biometal Science Laboratory RIKEN Harima Institute at SPring-8 1-1-1 Kouto, Sayo-cho Sayo-gun, Hyogo 679-5148 JAPAN Tel: (Office) +81-791-58-2838 (Direct) +81-791-58-0802 ext.7894 Fax: +81-791-58-2826 Email: tsk...@spring8.or.jp Web: http://www.riken.jp/biometal www.riken.jp/biometal or Https://sites.google.com/site/Kumarevel **
