Re: [ccp4bb] AutoXDS
Dear Marco, I did not know autoxds, but google suggests that it is a csh(uarrrgh)-script from SLAC. So as long as you have some c-Shell installed on your MAC, to script should execute. Maybe you can be more precise about what you mean by your last sentence to get more accurate help. Cheers, Tim On Wed, Mar 02, 2011 at 07:25:36PM +0100, Marco Lolicato wrote: Dear all, suggestion how to run autoxds on MAC OSX? I downloaded a script without success...do you know if are there others? Thanks, Marco -- -- 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] while on the subject of stereo
Dear Dave, Here come my five pence... I personally found stereo graphics useful in two cases. 1. When you first introduce students to biomolecular structure and/or biocrystallography. Showing stereo certainly helps 'building up' the initial fascination, which is very important of course. But since we do not have a classroom stereo setup, I am just speaking of seating a new/prospective (graduate) student in front of a stereo workstation. 2. When one performs more difficult tasks while doing research (although it was not your question). This includes building difficult regions in poor/low resolution maps as already mentioned, but maybe even more importantly when trying to make sense of a difficult MR case and finally when dealing with protein docking. However: 1. In my experience, what at least the better students do is to look at a structure using a simple program like Swiss PDB Viewer or Rasmol and their 300 euro laptop. No arguments can persuade them to use the 3000 euro lab stereo setup -- because they can manage to see what they want to see by just rotating the molecule... 2. For both teaching purposes and publications, I remain an adept of printed stereo pairs. Get each of your students a 5 euro stereo viewer and give them a handout full of stereo pairs rather than mono images. The very important this is that, on paper, one can make notes and drawings. An active digestion of the teaching material (rather than passive starring at your screen) has been known to help efficient learning since long ago... I can summarize my view as follows: for /most/ purposes, you should be fine by using one of the two: simple mono graphics to achieve the 3D effect by rotation -- or printed stereo pairs. HTH Sergei Thanks for the comments, I do appreciate them. I guess we went off in a direction I wasn't thinking of - related to your personal like or dislike of stereo. What I am really looking for is an answer to a simple question in that is stereo a nice thing from a pedagogy standpoint for showing students complex biomolecules. I am in a chemistry department - undergraduate only. We focus on 3-dimensional shape and the importance of shape of chemical function/reactivity/etc... With small molecules (PF5, etc...), it's easy to see how shape works by simply rotating the molecule. The molecules are small enough, the concept of 3D can be visualized easily in these systems. Furthermore, they can make a simple model using your standard organic or inorganic model kit, no worries. Now, bring in a huge protein, or a protein-protein complex. The issue of 3Dness becomes fuzzier. It's not so easy to see which hydrogen will get plucked off during a chemical reaction, even with careful zooming and mouse manipulation. So my question still is, how many of you feel stereo is important from a pedagogy standpoint (not looking at maps, just structures that are huge and complex). Is it something that we need to try to bring to the classroom, or is it just a cool toy like the 3D TV that hopefully is going nowhere and will soon fade out like the viewmaster of old. I know a large percentage of people cannot see stereo (at least the way we present it), and so it isn't for everybody. But, does it help, and if so, does it help when done in a huge classroom or when put on an individual screen. Has anybody tried to assess this (there's a horrible word for you). That's what I was wondering about. Presenting the stereo is a different issue (how is that done), but I think there are lots of avenues for that depending on your particular situation. Thanks again Dave
Re: [ccp4bb] Problem with refinement and positive electron density
I think you have been caught by a new REFMAC feature which tries to design its own TLS groups including linked H2Os and ligands. Check your tls output records and see what it has clustered into a group.. I am not sure how to disable this - at times I want to override any automatic selection.. Eleanor On 03/01/2011 07:48 PM, Judith Reeks wrote: Dear All, Thank you for your suggestions. Many of you asked what the occupancies were in the region and they were all one, so partial occupancy was not the problem. I was using TLS restraints during the refinement when this problem happened. Given the suggestions that TLS may be a problem and that might be causing the low B-factors, I went back and re-ran the refinement without TLS and the problem disappeared. Then I submitted my latest file to the TLSMD server for new restraints and the next round of refinement got rid of the problem. The B-factors increased to normal levels (~15 compared to ~5 before) so it seems to have done the trick. Thank you to everybody for their help, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews On 01/03/2011 17:28, Mark Robien wrote: Hmm - kinda interesting In addition to the sorts of things suggested by Mark van Raaij, older versions of Refmac were prone to have a phenomenon with B factors - once they get over a certain level, the algorithm has a very hard time bring them back down, even when the data suggests it. I've usually seen it with much higher B factors than you seem to have here - for example, loops where the B's are actually 40-60, but previous rounds of refinement have the B factors 60-80 or higher. Judging from the coot screen the residues you are focusing on, I doubt that is the answer (unless you also have a TLS model, in which case I'd wonder). If you do have TLS - well, things get more complicated; for example, is this the edge of a TLS domain? Nonetheless, you could try the solution for the problem that I describe - which is to reset all the B factors to a (very) low B factor - maybe even as low as 2.0 (lower?), and then another round of refmac - with a sufficient number of cycles - will re-refine the B (and xyz), thus escaping the region of refinement space that has a very weak gradient. A variant approach that might be appropriate - similarly reset the B for the (?small) region of your model that has the problem. Sadly, it's been awhile since I did any refinement myself - but the uppsala suite had some of the nicer tools for resetting B within pdb files, without having to do it manually (ugh - not appealing) - or writing an adhoc awk script (a very easy alternative, if you're familiar enough). Mark Robien On 3/1/2011 10:32 AM, Judith Reeks wrote: Dear All, I am currently refining a structure using the latest experimental version of Refmac (5.6) and there seems to be a problem with my Fo-Fc map. There is a region where I have fitted residues to the electron density but after refinement there is still positive electron density assigned to the region despite the fact that residues fit the electron density (see link below for a screenshot). Multiple rounds of refinement have yet to get rid of this problem. I have checked the PDB file and there does not appear to be any problems with this region. Has anybody seen something like this before? http://i1083.photobucket.com/albums/j382/jreeks/Screenshot2011-03-01at1613402.png Regards, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews
Re: [ccp4bb] Is there any program for specifically calculating Rvalue in CCP4
On 03/03/2011 06:13 AM, Ting-Wei Jiang wrote: Dear all experts, I'm trying to calculate R-value (and free R) specifically which is between data and the modified structure(refined by myself without help from any program).I've looked the program for calculating a long while.Actually,I found one named Rsearch (CCP4 supported).Nevertheless,I cant find Rsearch in CCP4 package. Could anyone direct me on how to get this value? thanks in advance. : TingWei Rsearch was a molecular replacement search program which calculated Rvalues for many positions in the unit cell. Not what you need. If you have a model and observed data the simplest way to get an Rfactor is to run 0 cycles of REFMAC. This will give you the Rfactors for your model without applying any shifts. It will adjust the scale and Bfactors to get the best ft though. If you already have a file containinf Fobs SigFobs and Fc rstats will calculate Rfactors. It is too old to consider Free Rfactors though All the best Eleanor
[ccp4bb] Scientific Programmer Position
Dear All, Please find below an announcement for a Scientic Programmer on the PROXIMA 2 beamline at Synchrotron SOLEIL. The position is a 2-year contract suitable for a PXer with strong computing instrumentation skills or a Computing Engineer. http://www.synchrotron-soleil.fr/images/File/soleil/DivisionAdministration/Personnel/SOLEIL_postDoc_PROXIMA_2.pdf http://www.synchrotron-soleil.fr/images/File/soleil/DivisionAdministration/Personnel/SOLEIL_postDoc_PROXIMA_2.pdf Kind regards, William SHEPARD PROXIMA 2 Beamline Responsible
[ccp4bb] I/sigmaI of 3.0 rule
Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
No - and I dont think it is accepted practice now either.. I often use I/SigI 1.5 for refinement.. Look at your Rfactor plots from REFMAC - if they look reasonable at higher resolution use the data Eleanor On 03/03/2011 11:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
Roberto, The reviewer's request is complete nonsense. The problem is how to best and politely respond so as not to prevent the paper from being accepted. Best would be to have educated editors who could simply tell you to ignore that request. Since this issue comes up quite often still, I think we all should come up with a canned response to such a request. One way to approach this issue is to avoid saying something like the structure has been refined to 2.2Å resolution, but instead say has been refined using data to a resolution of 2.2Å., or even has been refined using data with an I/sigmaI 1.5 (or whatever). Next could be to point out that even data with an I/sigmaI of 1 can contain information (I actually don't have a good reference for this, but I'm sure someone else can provide one'), and inclusion of such data can improve refinement stability and speed of convergence (not really important in a scientific sense, though). The point is that all of your data combined result in a structure with a certain resolution, pretty much no matter what high-resolution limits you choose (I/sigmaI of 0.5, 1.0, or 1.5). As long as you don't portrait your structure of having a resolution corresponding to the resolution of the high-resolution limit of your data, you should be fine. Now, requesting to toss out data with I/sigmaI of 3 simply reduces the resolution of your structure. You could calculate two electron density maps and show that your structure does indeed improve when including data with /sigmaI of 3. One criterion could be to use the optical resolution of the structure. Hope that helps. Best, MM On Mar 3, 2011, at 6:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
Dear Roberto, As indicated by others in reply to you the current best practice in protein crystallography is not a rigid application of such a cut off criterion. This is because there is such a diverse range of crystal qualities. However in chemical crystallography where the data quality from such crystals is more homogeneous such a rule is more often required notably as a guard against 'fast and loose' data collection which may occur (to achieve a very high throughput). As an Editor myself, whilst usually allowing the authors' chosen resolution cut off, I will insist on the data table saying in a footnote the diffraction resolution where I/sig(I) crosses 2.0 and/or, if relevant, where DeltaAnom/sig(DeltaAnom) crosses 1.0. A remaining possible contentious point with a submitting author is where the title of a paper may claim a diffraction resolution that in fact cannot really be substantiated. Best wishes, Yours sincerely, John On Thu, Mar 3, 2011 at 11:29 AM, Roberto Battistutta roberto.battistu...@unipd.it wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it -- Professor John R Helliwell DSc
Re: [ccp4bb] Problem with refinement and positive electron density
Dear Eleanor, I don't think that was the case. I add tlsd waters exclude to a refinement which as I understand it should prevent that. Also, I checked the output files and the TLS groups only involve my protein, waters and ligands aren't included anywhere. I can't explain why my TLS restraints were giving me problems. Regards, Judith On 03/03/2011 10:49, Eleanor Dodson wrote: I think you have been caught by a new REFMAC feature which tries to design its own TLS groups including linked H2Os and ligands. Check your tls output records and see what it has clustered into a group.. I am not sure how to disable this - at times I want to override any automatic selection.. Eleanor On 03/01/2011 07:48 PM, Judith Reeks wrote: Dear All, Thank you for your suggestions. Many of you asked what the occupancies were in the region and they were all one, so partial occupancy was not the problem. I was using TLS restraints during the refinement when this problem happened. Given the suggestions that TLS may be a problem and that might be causing the low B-factors, I went back and re-ran the refinement without TLS and the problem disappeared. Then I submitted my latest file to the TLSMD server for new restraints and the next round of refinement got rid of the problem. The B-factors increased to normal levels (~15 compared to ~5 before) so it seems to have done the trick. Thank you to everybody for their help, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews On 01/03/2011 17:28, Mark Robien wrote: Hmm - kinda interesting In addition to the sorts of things suggested by Mark van Raaij, older versions of Refmac were prone to have a phenomenon with B factors - once they get over a certain level, the algorithm has a very hard time bring them back down, even when the data suggests it. I've usually seen it with much higher B factors than you seem to have here - for example, loops where the B's are actually 40-60, but previous rounds of refinement have the B factors 60-80 or higher. Judging from the coot screen the residues you are focusing on, I doubt that is the answer (unless you also have a TLS model, in which case I'd wonder). If you do have TLS - well, things get more complicated; for example, is this the edge of a TLS domain? Nonetheless, you could try the solution for the problem that I describe - which is to reset all the B factors to a (very) low B factor - maybe even as low as 2.0 (lower?), and then another round of refmac - with a sufficient number of cycles - will re-refine the B (and xyz), thus escaping the region of refinement space that has a very weak gradient. A variant approach that might be appropriate - similarly reset the B for the (?small) region of your model that has the problem. Sadly, it's been awhile since I did any refinement myself - but the uppsala suite had some of the nicer tools for resetting B within pdb files, without having to do it manually (ugh - not appealing) - or writing an adhoc awk script (a very easy alternative, if you're familiar enough). Mark Robien On 3/1/2011 10:32 AM, Judith Reeks wrote: Dear All, I am currently refining a structure using the latest experimental version of Refmac (5.6) and there seems to be a problem with my Fo-Fc map. There is a region where I have fitted residues to the electron density but after refinement there is still positive electron density assigned to the region despite the fact that residues fit the electron density (see link below for a screenshot). Multiple rounds of refinement have yet to get rid of this problem. I have checked the PDB file and there does not appear to be any problems with this region. Has anybody seen something like this before? http://i1083.photobucket.com/albums/j382/jreeks/Screenshot2011-03-01at1613402.png Regards, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews
Re: [ccp4bb] Is there any program for specifically calculating Rvalue in CCP4
On Thu, 2011-03-03 at 14:13 +0800, Ting-Wei Jiang wrote: I'm trying to calculate R-value (and free R) specifically which is between data and the modified structure(refined by myself without help from any program). At long last, someone escaped the tyranny and oppression of the refinement programs and deciphered the message hidden within the data by using the mystical power known to the ancients as X-ray vision. Paging Dan Brown. On a serious note, if you have the Fo and Fc, the calculation is rather trivial (if that is the case, let me know and I'll send you a little piece of code that does that). However, I suspect that all you have is the model (xyzb for every atom) and Fo. In which case there are many ways to do the calulation. - sfcheck - refmac with 0 rounds of refinement (already mentioned by Eleanor) - CNS script model_stats.inp - clipper libs - phenix.fmodel will probably output it too or maybe there is some phenix.get_me_my_damn_rfactor or such The problem you'll have is that values will be different with different programs. Given that your model is exactly the same and presumably the atomic scattering factors used by these programs are the same, the few likely reasons are various scaling operations applied. IMHO, the main source of difference should be the way one treats the bulk solvent correction. HTH, and yes, the first paragraph is meant to be a joke. Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
On Thu, 2011-03-03 at 12:29 +0100, Roberto Battistutta wrote: Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? There is none. Did editor ask you to follow this suggestion? I wonder if there is anyone among the subscribers of this bb who would come forward and support this I/sigmaI 3.0 claim. What was your I/sigma, by the way? I almost always collect data to I/sigma=1, which has the downside of generating somewhat higher R-values. Shall I, according to this reviewer, retract/amend every single one of them? What a mess. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] while on the subject of stereo
On Wed, 2011-03-02 at 19:23 -0500, David Roberts wrote: What I am really looking for is an answer to a simple question in that is stereo a nice thing from a pedagogy standpoint for showing students complex biomolecules. Of course it is. Exactly how much excitement it generates among the millennials with their iThings is hard to gauge, but it definitely has greater potential as a teaching tool than the formula for phased translation function. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
There seem to be quite a few rule followers out there regarding resolution cutoffs. One that I have encountered several times is reviewers objecting to high Rsym values (say 60-80% in the last shell), which may be even worse than using some fixed value of I/sigI. On 3/3/11 9:55 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2011-03-03 at 12:29 +0100, Roberto Battistutta wrote: Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? There is none. Did editor ask you to follow this suggestion? I wonder if there is anyone among the subscribers of this bb who would come forward and support this I/sigmaI 3.0 claim. What was your I/sigma, by the way? I almost always collect data to I/sigma=1, which has the downside of generating somewhat higher R-values. Shall I, according to this reviewer, retract/amend every single one of them? What a mess. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] stuck with COOT installation in openSUSE 11.3
On 02/28/2011 09:39 PM, Hena Dutta wrote: I could not open the COOT GUI after installing either from 'coot-0.6.1-binary-Linux-x86_64-centos-5-gtk2.tar.gz' or from 'coot-0.6.2-pre-1-revision-3205-binary-Linux-x86_64-centos-5-gtk2.tar.gz' I have managed to build a coot pre-release for OpenSuse 11.3 64-bit. http://lmb.bioch.ox.ac.uk/coot/software/binaries/pre-releases/coot-0.6.2-pre-1-revision-3405-binary-Linux_x86_64-OpenSuse-11.3-gtk2.tar.gz It should just work. (This is the non-python version, if you want some fun, try compiling python 2.6 on OpenSuse 11.3 either 1) without the system readline or 2) with a by-hand installed readline. Grrr... wretched thing - the only people who like python are the ones who don't have to compile it... end-rant) Paul.
Re: [ccp4bb] I/sigmaI of 3.0 rule
For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. As a reviewer, when I have to evaluate a manuscript where very high Rsym values are quoted, I have no way of knowing how the high resolution cutoff was set. So I simply suggest to the authors to double check this cutoff, in order to ensure that the high resolution limit really corresponds to high resolution data and not to noise. But I certainly do not make statements such as this one. I have seen cases where, using this rule to decide on the high resolution limit, the Rsym in the high resolution bin is well below 50% and cases where it is much higher. Like 65%, 70% (0.65, 0.7 if you prefer). So, in my opinion, there is no fixed rule as to what the acceptable Rsym value in the highest resolution shell should be. Fred. Van Den Berg, Bert wrote: There seem to be quite a few “rule” followers out there regarding resolution cutoffs. One that I have encountered several times is reviewers objecting to high Rsym values (say 60-80% in the last shell), which may be even worse than using some fixed value of I/sigI. On 3/3/11 9:55 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2011-03-03 at 12:29 +0100, Roberto Battistutta wrote: Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? There is none. Did editor ask you to follow this suggestion? I wonder if there is anyone among the subscribers of this bb who would come forward and support this I/sigmaI 3.0 claim. What was your I/sigma, by the way? I almost always collect data to I/sigma=1, which has the downside of generating somewhat higher R-values. Shall I, according to this reviewer, retract/amend every single one of them? What a mess. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
As mentioned there is no I/sigmaI rule. Also you need to specify (and correctly calculate) I/sigmaI and not I/sigmaI. A review of similar articles in the same journal will show what is typical for the journal. I think you will find that the I/sigmaI cutoff varies. This information can be used in your response to the reviewer as in, A review of actual published articles in the Journal shows that 75% (60 out of 80) used an I/sigmaI cutoff of 2 for the resolution of the diffraction data used in refinement. We respectfully believe that our cutoff of 2 should be acceptable. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roberto Battistutta Sent: Thursday, March 03, 2011 5:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] I/sigmaI of 3.0 rule Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
My preferred criterion is the half-dataset correlation coefficient output by Scala (an idea stolen from the EM guys): I tend to cut my data where this falls to not less than 0.5. The good thing about this is that it is independent of the vagaries of I/sigma (or rather of the SD estimation) and has a more intuitive cutoff point than Rmeas (let alone Rmerge). It probably doesn't work well at low multiplicity and there is always a problem with anisotropy (I intend to do anisotropic analysis in future) That said, the exact resolution cut-off is not really important: if you refine look at maps at say 2.6A vs. 2.5A (if that's around the potential cutoff), there is probably little significant difference Phil On 3 Mar 2011, at 15:34, Jim Pflugrath wrote: As mentioned there is no I/sigmaI rule. Also you need to specify (and correctly calculate) I/sigmaI and not I/sigmaI. A review of similar articles in the same journal will show what is typical for the journal. I think you will find that the I/sigmaI cutoff varies. This information can be used in your response to the reviewer as in, A review of actual published articles in the Journal shows that 75% (60 out of 80) used an I/sigmaI cutoff of 2 for the resolution of the diffraction data used in refinement. We respectfully believe that our cutoff of 2 should be acceptable. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roberto Battistutta Sent: Thursday, March 03, 2011 5:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] I/sigmaI of 3.0 rule Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
I think this suppression of high resolution shells via I/sigI cutoffs is partially attributable to a conceptual misunderstanding of what these (darn) R-values mean in refinement versus data merging. In refinement, even a random atom structure follows the Wilson distribution, and therefore, even a completely wrong non-centrosymmetric structure will not - given proper scaling - give an Rf of more than 59%. There is no such limit for the basic linear merging R. However, there is a simple relation between I/sigI and R-merge (provided no other indecency has been done to the data). It simply is (BMC) Rm=0.8/I/sigI. I.e. for I/sigI -0.8 you get 100%, for 2 we obtain 40%, which, interpreted as Rf would be dreadful, but for I/sigI 3, we get Rm=0.27, and that looks acceptable for an Rf (or uninformed reviewer). Btw, I also wish to point out that the I/sig cutoffs are not exactly the cutoff criterion for anomalous phasing, a more direct measure is a signal cutoff such as delF/sig(delF); George I believe uses 1.3 for SAD. Interestingly, in almost all structures I played with, delF/sig(delF) for both, noise in anomalous data or no anomalous scatterer present, the anomalous signal was 0.8. I havent figured out yet or proved the statistics and whether this is generally true or just numerology... And, the usual biased rant - irrespective of Hamilton tests, nobody really needs these popular unweighted linear residuals which shall not be named, particularly on F. They only cause trouble. Best regards, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - Structural Biology is the practice of crystallography without a license. - -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bart Hazes Sent: Thursday, March 03, 2011 7:08 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule There seems to be an epidemic of papers with I/Sigma 3 (sometime much larger). In fact such cases have become so frequent that I fear some people start to believe that this is the proper procedure. I don't know where that has come from as the I/Sigma ~ 2 criterion has been established long ago and many consider that even a tad conservative. It simply pains me to see people going to the most advanced synchrotrons to boost their highest resolution data and then simply throw away much of it. I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas - High I/Sigma cutoffs are normal for (S/M)AD data sets where a more strict focus on data quality is needed. Perhaps some people have started to think this is the norm. - For some dataset Rsym goes up strongly while I/SigI is still reasonable. I personally believe this is due to radiation damage which affects Rsym (which compares reflections taken after different amounts of exposure) much more than I/SigI which is based on individual reflections. A good test would be to see if processing only the first half of the dataset improves Rsym (or better Rrim) - Most detectors are square and if the detector is too far from the crystal then the highest resolution data falls beyond the edges of the detector. In this case one could, and should, still process data into the corners of the detector. Data completeness at higher resolution may suffer but each additional reflection still represents an extra restraint in refinement and a Fourier term in the map. Due to crystal symmetry the effect on completeness may even be less than expected. Bart On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it -- 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:
Re: [ccp4bb] I/sigmaI of 3.0 rule
On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote: For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. Fred, while your procedure is definitely more sophisticated than what I do, let me point out that the Rsym is genuinely a bad measure for this, as it depends strongly on redundancy. Does more robust measures (e.g. Rpim) show similar inflexion? I suspect it will at least shift towards higher resolution. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
On Thu, 2011-03-03 at 08:08 -0700, Bart Hazes wrote: I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas It may also be related to what I feel is recent revival of the significance of the R-values in general. Lower resolution cutoffs in this context improve the R-values, which is (incorrectly) perceived as model improvement. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
Does the position of this inflection point depend on the redundancy? Maybe it does not; for high-redundancy data one would simply get a much higher corresponding Rsym. On 3/3/11 11:13 AM, Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote: For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. Fred, while your procedure is definitely more sophisticated than what I do, let me point out that the Rsym is genuinely a bad measure for this, as it depends strongly on redundancy. Does more robust measures (e.g. Rpim) show similar inflexion? I suspect it will at least shift towards higher resolution. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
On Thu, 2011-03-03 at 09:34 -0600, Jim Pflugrath wrote: As mentioned there is no I/sigmaI rule. Also you need to specify (and correctly calculate) I/sigmaI and not I/sigmaI. A review of similar articles in the same journal will show what is typical for the journal. I think you will find that the I/sigmaI cutoff varies. This information can be used in your response to the reviewer as in, A review of actual published articles in the Journal shows that 75% (60 out of 80) used an I/sigmaI cutoff of 2 for the resolution of the diffraction data used in refinement. We respectfully believe that our cutoff of 2 should be acceptable. Jim, Excellent point. Such statistics would be somewhat tedious to gather though, does anyone know if I/sigma stats are available for the whole PDB somewhere? On your first point though - why is one better than the other? My experimental observation is while the two differ significantly at low resolution (what matters, of course, is I/sigma itself and not the resolution per se), at high resolution where the cutoff is chosen they are not that different. And since the cutoff value itself is rather arbitrarily chosen, then why I/sigma is better than I/sigma? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
related to what I feel is recent revival of the significance of the R-values because it's so handy to have one single number to judge a highly complex nonlinear multivariate barely determined regularized problem! Just as easy as running a gel! Best BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Thursday, March 03, 2011 8:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule On Thu, 2011-03-03 at 08:08 -0700, Bart Hazes wrote: I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas It may also be related to what I feel is recent revival of the significance of the R-values in general. Lower resolution cutoffs in this context improve the R-values, which is (incorrectly) perceived as model improvement. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
Hi, I don't think XDS generates an Rpim value, does it? The XDS CORRECT strep provides the old fashioned Rsym (R-FACTOR) plus R-meas and Rmrgd-F. The curves look all the same though Fred. Ed Pozharski wrote: On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote: For myself, I decide on the high resolution cutoff by looking at the Rsym vs resolution curve. The curve rises, and for all data sets I have processed (so far) there is a break in the curve and the curve shoots up. To near vertical. This inflexion point is where I decide to place the high resolution cutoff, I never look at the I/sigma(I) values nor at the Rsym in the high resolution shell. Fred, while your procedure is definitely more sophisticated than what I do, let me point out that the Rsym is genuinely a bad measure for this, as it depends strongly on redundancy. Does more robust measures (e.g. Rpim) show similar inflexion? I suspect it will at least shift towards higher resolution. Cheers, Ed.
Re: [ccp4bb] Is there any program for specifically calculating Rvalue in CCP4
Hi, - phenix.fmodel will probably output it too or maybe there is some phenix.get_me_my_damn_rfactor or such as Eric pointed out earlier, the command phenix.model_vs_data model.pdb data.mtz will do exactly this. Pavel. P.S.: phenix.fmodel is the tool to compute total model structure factor, and this one phenix.get_me_my_damn_rfactor does not exist -:)
Re: [ccp4bb] I/sigmaI of 3.0 rule
Discussions of I/sigma(I) or less-than cutoffs have been going on for at least 35 years. For example, see Acta Cryst. (1975) B31, 1507-1509. I was taught by my elders (mainly Lyle Jensen) that less-than cutoffs came into use when diffractometers replaced film methods for small molecule work, i.e., 1960s. To compare new and old structures, they needed some criterion for the electronic measurements that would correspond to the fog level on their films. People settled on 2 sigma cutoffs (on I which mean 4 sigma on F), but subsequently, the cutoffs got higher and higher, as people realized they could get lower and lower R values by throwing away the weak reflections. I'm unaware of any statistical justification for any cutoff. The approach I like the most is to refine on Fsquared and use every reflection. Error estimates and weighting schemes should take care of the noise. Ron On Thu, 3 Mar 2011, Ed Pozharski wrote: On Thu, 2011-03-03 at 09:34 -0600, Jim Pflugrath wrote: As mentioned there is no I/sigmaI rule. Also you need to specify (and correctly calculate) I/sigmaI and not I/sigmaI. A review of similar articles in the same journal will show what is typical for the journal. I think you will find that the I/sigmaI cutoff varies. This information can be used in your response to the reviewer as in, A review of actual published articles in the Journal shows that 75% (60 out of 80) used an I/sigmaI cutoff of 2 for the resolution of the diffraction data used in refinement. We respectfully believe that our cutoff of 2 should be acceptable. Jim, Excellent point. Such statistics would be somewhat tedious to gather though, does anyone know if I/sigma stats are available for the whole PDB somewhere? On your first point though - why is one better than the other? My experimental observation is while the two differ significantly at low resolution (what matters, of course, is I/sigma itself and not the resolution per se), at high resolution where the cutoff is chosen they are not that different. And since the cutoff value itself is rather arbitrarily chosen, then why I/sigma is better than I/sigma? Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule- do not underestimate gels
Well BR, do not underestimate complexity of running a gel! There are even more harsh referees comments on gel appearance and quality than comments on cutting data based on R,RF and sigmaI :-) Especially when one is trying to penetrate into prestigious journals... Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Mar 3, 2011, at 18:38 , Bernhard Rupp (Hofkristallrat a.D.) wrote: related to what I feel is recent revival of the significance of the R-values because it's so handy to have one single number to judge a highly complex nonlinear multivariate barely determined regularized problem! Just as easy as running a gel! Best BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Thursday, March 03, 2011 8:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule On Thu, 2011-03-03 at 08:08 -0700, Bart Hazes wrote: I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas It may also be related to what I feel is recent revival of the significance of the R-values in general. Lower resolution cutoffs in this context improve the R-values, which is (incorrectly) perceived as model improvement. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONS COMPLET R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 10.06 5509 304 364 83.5% 3.0% 4.4% 5509 63.83 3.1% 1.0% 11% 0.652 173 7.12 11785 595 595 100.0% 3.5% 4.8% 11785 59.14 3.6% 1.4% -10% 0.696 414 5.81 15168 736 736 100.0% 5.0% 5.6% 15168 51.88 5.1% 1.8% -9% 0.692 561 5.03 17803 854 854 100.0% 5.5% 5.7% 17803 50.02 5.6% 2.2% -10% 0.738 675 4.50 20258 964 964 100.0% 5.1% 5.4% 20258 52.61 5.3% 2.1% -16% 0.710 782 4.11 22333 1054 1054 100.0% 5.6% 5.7% 22333 50.89 5.8% 2.0% -16% 0.705 878 3.80 23312 1137 1137 100.0% 7.0% 6.6% 23312 42.95 7.1% 3.0% -13% 0.770 952 3.56 25374 1207 1208 99.9% 7.6% 7.3% 25374 40.56 7.8% 3.4% -18% 0.739 1033 3.35 27033 1291 1293 99.8% 9.7% 9.2% 27033 33.73 10.0% 4.1% -12% 0.765 1107 3.18 29488 1353 1353 100.0% 11.6% 11.6% 29488 28.16 11.9% 4.4% -7% 0.750 1176 3.03 31054 1419 1419 100.0% 15.7% 15.9% 31054 21.77 16.0% 6.9% -9% 0.741 1243 2.90 32288 1478 1478 100.0% 21.1% 21.6% 32288 16.99 21.6% 9.2% -6% 0.745 1296 2.79 33807 1542 1542 100.0% 28.1% 28.8% 33807 13.07 28.8% 12.9% -2% 0.783 1361 2.69 34983 1604 1604 100.0% 37.4% 38.7% 34983 9.95 38.3% 17.2% -2% 0.743 1422 2.60 35163 1653 1653 100.0% 48.8% 48.0% 35163 8.03 50.0% 21.9% -6% 0.754 1475 2.52 36690 1699 1699 100.0% 54.0% 56.0% 36690 6.98 55.3% 25.9% 0% 0.745 1517 2.44 37751 1757 1757 100.0% 67.9% 70.4% 37751 5.61 69.5% 32.5% -5% 0.733 1577 2.37 38484 1798 1799 99.9% 82.2% 84.5% 38484 4.72 84.2% 36.5% 2% 0.753 1620 2.31 39098 1842 1842 100.0% 91.4% 94.3% 39098 4.19 93.7% 43.7% -3% 0.744 1661 2.25 38809 1873 1923 97.4% 143.4% 139.3% 38809 2.84 147.1% 69.8% -2% 0.693 1696 total 556190 26160 26274 99.6% 11.9% 12.2% 556190 21.71 12.2% 9.7% -5% 0.739 22619 Bernhard Rupp (Hofkristallrat a.D.) wrote: I think this suppression of high resolution shells via I/sigI cutoffs is partially attributable to a conceptual misunderstanding of what these (darn) R-values mean in refinement versus data merging. In refinement, even a random atom structure follows the Wilson distribution, and therefore, even a completely wrong non-centrosymmetric structure will not - given proper scaling - give an Rf of more than 59%. There is no such limit for the basic linear merging R. However, there is a simple relation between I/sigI and R-merge (provided no other indecency has been done to the data). It simply is (BMC) Rm=0.8/I/sigI. I.e. for I/sigI -0.8 you get 100%, for 2 we obtain 40%, which, interpreted as Rf would be dreadful, but for I/sigI 3, we get Rm=0.27, and that looks acceptable for an Rf (or uninformed reviewer). Btw, I also wish to point out that the I/sig cutoffs are not exactly the cutoff criterion for anomalous phasing, a more direct measure is a signal cutoff such as delF/sig(delF); George I believe uses 1.3 for SAD. Interestingly, in almost all structures I played with, delF/sig(delF) for both, noise in anomalous data or no anomalous scatterer present, the anomalous signal was 0.8. I haven’t figured out yet or proved the statistics and whether this is generally true or just numerology... And, the usual biased rant - irrespective of Hamilton tests, nobody really needs these popular unweighted linear residuals which shall not be named, particularly on F. They only cause trouble. Best regards, BR - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 b...@ruppweb.org hofkristall...@gmail.com http://www.ruppweb.org/ - Structural Biology is the practice of crystallography without a license. - -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bart Hazes Sent: Thursday, March 03, 2011 7:08 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule There seems to be an epidemic of papers with I/Sigma 3 (sometime much larger). In fact such cases have become so frequent that I fear some people start to believe that this is the proper procedure. I don't know where that has come from as the I/Sigma ~ 2 criterion has been established long ago and many consider that even a tad conservative. It simply pains me to see people going to the most advanced synchrotrons to boost their highest resolution data and then simply throw away much of it. I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas - High I/Sigma cutoffs are normal for (S/M)AD data sets where a more strict focus on data quality is needed. Perhaps some people have started to think this is the norm. - For some dataset Rsym goes up strongly while I/SigI
Re: [ccp4bb] I/sigmaI of 3.0 rule
I take the point about a tendency in those days to apply sigma cutoffs to get lower R values, which were erroneously expected to indicate better structures. I wonder how many of us remember this paper by Arnberg et al (1979) Acta Cryst A35, 497-499, where it is shown for (small molecule) structures that had been refined with only reflections I3*sigma(I) that the models were degraded by leaving out weak data (although the R factors looked better of course). Arnberg et al took published structures and showed the refined models got better when the weak data were included. The best bit, I think, was when they went on to demonstrate successful refinement of a structure using ONLY the weak data where I3*sigma(I) and ignoring all the strong ones. This shows, as was alluded to earlier in the discussion, that a weak reflection puts a powerful constraint on a refinement, especially if there are other stronger reflections in the same resolution range. --- | Simon E.V. Phillips | --- | Director, Research Complex at Harwell (RCaH)| | Rutherford Appleton Laboratory | | Harwell Science and Innovation Campus | | Didcot | | Oxon OX11 0FA | | United Kingdom | | Email: simon.phill...@rc-harwell.ac.uk | | Tel: +44 (0)1235 567701 | |+44 (0)1235 567700 (sec) | |+44 (0)7884 436011 (mobile) | | www.rc-harwell.ac.uk| --- | Astbury Centre for Structural Molecular Biology | | Institute of Molecular and Cellular Biology | | University of LEEDS | | LEEDS LS2 9JT | | United Kingdom | | Email: s.e.v.phill...@leeds.ac.uk | | Tel: +44 (0)113 343 3027 | | WWW: http://www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SEVP | ---
Re: [ccp4bb] I/sigmaI of 3.0 rule
I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it DETECTOR_SU SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE = -3.0 AS FUNCTION OF RESOLUTION RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR R-FACTOR COMPARED I/SIGMA R-meas Rmrgd-F Anomal SigAno Nano LIMIT OBSERVED UNIQUE POSSIBLE OF DATA observed expected Corr 10.065509 304 364 83.5% 3.0% 4.4% 5509 63.83 3.1% 1.0%11% 0.652 173 7.12 11785 595 595 100.0% 3.5% 4.8% 11785 59.14 3.6% 1.4% -10% 0.696 414 5.81 15168 736 736 100.0% 5.0% 5.6% 15168 51.88 5.1% 1.8%-9% 0.692 561 5.03 17803 854 854 100.0% 5.5% 5.7% 17803 50.02 5.6% 2.2% -10% 0.738 675 4.50 20258 964 964 100.0% 5.1% 5.4% 20258 52.61 5.3% 2.1% -16% 0.710 782 4.11 223331054 1054 100.0% 5.6% 5.7% 22333 50.89 5.8% 2.0% -16% 0.705 878 3.80 233121137 1137 100.0% 7.0% 6.6% 23312 42.95 7.1% 3.0% -13% 0.770 952 3.56 253741207 1208 99.9% 7.6% 7.3% 25374 40.56 7.8% 3.4% -18% 0.7391033 3.35 270331291 1293 99.8% 9.7% 9.2% 27033 33.7310.0% 4.1% -12% 0.7651107 3.18 294881353 1353 100.0% 11.6% 11.6% 29488 28.1611.9% 4.4%-7% 0.7501176 3.03 310541419 1419 100.0% 15.7% 15.9% 31054 21.7716.0% 6.9%-9% 0.7411243 2.90 322881478 1478 100.0% 21.1% 21.6% 32288 16.9921.6% 9.2%-6% 0.7451296 2.79 338071542 1542 100.0% 28.1% 28.8% 33807 13.0728.8%12.9%-2% 0.7831361 2.69 349831604 1604 100.0% 37.4% 38.7% 349839.9538.3%17.2%-2% 0.7431422 2.60 351631653 1653 100.0% 48.8% 48.0% 351638.0350.0%21.9%-6% 0.7541475 2.52 366901699 1699 100.0% 54.0% 56.0% 366906.9855.3%25.9% 0% 0.7451517 2.44 377511757 1757 100.0% 67.9% 70.4% 377515.6169.5%32.5%-5% 0.7331577 2.37 384841798 1799 99.9% 82.2% 84.5% 384844.7284.2%36.5% 2% 0.7531620 2.31 390981842 1842 100.0% 91.4% 94.3% 390984.1993.7%43.7%-3% 0.7441661 2.25 388091873 1923 97.4% 143.4%139.3% 388092.84 147.1%69.8%-2% 0.6931696 total 556190 26160 26274 99.6% 11.9% 12.2% 556190 21.7112.2% 9.7%-5% 0.739 22619
Re: [ccp4bb] I/sigmaI of 3.0 rule- do not underestimate gels
there are even more harsh referees comments on gel appearance and quality than comments on cutting data based on R,RF and sigmaI :-) Especially when one is trying to penetrate into prestigious journals... Ok I repent. For improving gels there is the same excellent program, also useful for density modification - Photoshop ;-) Best, BR Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Mar 3, 2011, at 18:38 , Bernhard Rupp (Hofkristallrat a.D.) wrote: related to what I feel is recent revival of the significance of the R-values because it's so handy to have one single number to judge a highly complex nonlinear multivariate barely determined regularized problem! Just as easy as running a gel! Best BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Thursday, March 03, 2011 8:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule On Thu, 2011-03-03 at 08:08 -0700, Bart Hazes wrote: I don't know what has caused this wave of high I/Sigma threshold use but here are some ideas It may also be related to what I feel is recent revival of the significance of the R-values in general. Lower resolution cutoffs in this context improve the R-values, which is (incorrectly) perceived as model improvement. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] I/sigmaI of 3.0 rule
We should compile this discussion and send it as compulsive reading to journal editors...;-) Bert On 3/3/11 12:07 PM, Simon Phillips s.e.v.phill...@leeds.ac.uk wrote: I take the point about a tendency in those days to apply sigma cutoffs to get lower R values, which were erroneously expected to indicate better structures. I wonder how many of us remember this paper by Arnberg et al (1979) Acta Cryst A35, 497-499, where it is shown for (small molecule) structures that had been refined with only reflections I3*sigma(I) that the models were degraded by leaving out weak data (although the R factors looked better of course). Arnberg et al took published structures and showed the refined models got better when the weak data were included. The best bit, I think, was when they went on to demonstrate successful refinement of a structure using ONLY the weak data where I3*sigma(I) and ignoring all the strong ones. This shows, as was alluded to earlier in the discussion, that a weak reflection puts a powerful constraint on a refinement, especially if there are other stronger reflections in the same resolution range. --- | Simon E.V. Phillips | --- | Director, Research Complex at Harwell (RCaH)| | Rutherford Appleton Laboratory | | Harwell Science and Innovation Campus | | Didcot | | Oxon OX11 0FA | | United Kingdom | | Email: simon.phill...@rc-harwell.ac.uk | | Tel: +44 (0)1235 567701 | |+44 (0)1235 567700 (sec) | |+44 (0)7884 436011 (mobile) | | www.rc-harwell.ac.uk| --- | Astbury Centre for Structural Molecular Biology | | Institute of Molecular and Cellular Biology | | University of LEEDS | | LEEDS LS2 9JT | | United Kingdom | | Email: s.e.v.phill...@leeds.ac.uk | | Tel: +44 (0)113 343 3027 | | WWW: http://www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SEVP http://www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SEVP | ---
Re: [ccp4bb] I/sigmaI of 3.0 rule
When will we finally jettison Rsym/Rcryst/Rmerge? 1. Perhaps software developers should either not even calculate the number, or hide it somewhere obscure, and of course replacing it with a better R flavor? 2. Maybe reviewers should insist on other R's (Rpim etc) instead of Rmerge? JPK PS is this as quixotic as chucking the QWERTY keyboard, or using Esperanto? I don't think so! On Thu, Mar 3, 2011 at 11:07 AM, Simon Phillips s.e.v.phill...@leeds.ac.uk wrote: I take the point about a tendency in those days to apply sigma cutoffs to get lower R values, which were erroneously expected to indicate better structures. I wonder how many of us remember this paper by Arnberg et al (1979) Acta Cryst A35, 497-499, where it is shown for (small molecule) structures that had been refined with only reflections I3*sigma(I) that the models were degraded by leaving out weak data (although the R factors looked better of course). Arnberg et al took published structures and showed the refined models got better when the weak data were included. The best bit, I think, was when they went on to demonstrate successful refinement of a structure using ONLY the weak data where I3*sigma(I) and ignoring all the strong ones. This shows, as was alluded to earlier in the discussion, that a weak reflection puts a powerful constraint on a refinement, especially if there are other stronger reflections in the same resolution range. --- | Simon E.V. Phillips | --- | Director, Research Complex at Harwell (RCaH) | | Rutherford Appleton Laboratory | | Harwell Science and Innovation Campus | | Didcot | | Oxon OX11 0FA | | United Kingdom | | Email: simon.phill...@rc-harwell.ac.uk | | Tel: +44 (0)1235 567701 | | +44 (0)1235 567700 (sec) | | +44 (0)7884 436011 (mobile) | | www.rc-harwell.ac.uk | --- | Astbury Centre for Structural Molecular Biology | | Institute of Molecular and Cellular Biology | | University of LEEDS | | LEEDS LS2 9JT | | United Kingdom | | Email: s.e.v.phill...@leeds.ac.uk | | Tel: +44 (0)113 343 3027 | | WWW: http://www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SEVP | --- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Problem with refinement and positive electron density
On Thursday, March 03, 2011 05:10:02 am Judith Reeks wrote: Dear Eleanor, I don't think that was the case. I add tlsd waters exclude to a refinement which as I understand it should prevent that. Also, I checked the output files and the TLS groups only involve my protein, waters and ligands aren't included anywhere. I can't explain why my TLS restraints were giving me problems. Without looking at your files I can't be certain, but The usual problem is that refmac applies clipping limits to B factors. In the absence of TLS, this means that the minimum allowed B is (by default) 2.0 and the maximum is (I think) 200. So far. so good. But in the presence of a TLS model, these same clipping limits are applied to the _incremental_ Biso values, and that's just wrong. I.e., if the current set of TLS parameters over-estimates B for some particular atom, the density gradient will try to drive the incremental B value for that atom negative to compensate. But the program doesn't let that happen. The result is that the incremental B values bottom out at 2.0 and the refinement goes pear-shaped. I think the simplest fix for this would be to have refmac detect that it has happened and modify the current TLS parameters to shift all the predicted B values downward. That way the required incremental Biso become 0 and the problem is avoided. An alternative, but more difficult to implement, fix would be to let the incremental B values go negative. The paired *.pdb and *.tlsin files output by the TLSMD server specifically avoid this problem: the TLS parameters are chosen such that the incremental B values required to best reproduce your input model are always positive. But as you continue refinement this criteria may gradually be lost, and as noted above refmac doesn't currently detect or fix this for you. Ethan Regards, Judith On 03/03/2011 10:49, Eleanor Dodson wrote: I think you have been caught by a new REFMAC feature which tries to design its own TLS groups including linked H2Os and ligands. Check your tls output records and see what it has clustered into a group.. I am not sure how to disable this - at times I want to override any automatic selection.. Eleanor On 03/01/2011 07:48 PM, Judith Reeks wrote: Dear All, Thank you for your suggestions. Many of you asked what the occupancies were in the region and they were all one, so partial occupancy was not the problem. I was using TLS restraints during the refinement when this problem happened. Given the suggestions that TLS may be a problem and that might be causing the low B-factors, I went back and re-ran the refinement without TLS and the problem disappeared. Then I submitted my latest file to the TLSMD server for new restraints and the next round of refinement got rid of the problem. The B-factors increased to normal levels (~15 compared to ~5 before) so it seems to have done the trick. Thank you to everybody for their help, Judith Reeks ja...@st-andrews.ac.uk School of Chemistry University of St Andrews On 01/03/2011 17:28, Mark Robien wrote: Hmm - kinda interesting In addition to the sorts of things suggested by Mark van Raaij, older versions of Refmac were prone to have a phenomenon with B factors - once they get over a certain level, the algorithm has a very hard time bring them back down, even when the data suggests it. I've usually seen it with much higher B factors than you seem to have here - for example, loops where the B's are actually 40-60, but previous rounds of refinement have the B factors 60-80 or higher. Judging from the coot screen the residues you are focusing on, I doubt that is the answer (unless you also have a TLS model, in which case I'd wonder). If you do have TLS - well, things get more complicated; for example, is this the edge of a TLS domain? Nonetheless, you could try the solution for the problem that I describe - which is to reset all the B factors to a (very) low B factor - maybe even as low as 2.0 (lower?), and then another round of refmac - with a sufficient number of cycles - will re-refine the B (and xyz), thus escaping the region of refinement space that has a very weak gradient. A variant approach that might be appropriate - similarly reset the B for the (?small) region of your model that has the problem. Sadly, it's been awhile since I did any refinement myself - but the uppsala suite had some of the nicer tools for resetting B within pdb files, without having to do it manually (ugh - not appealing) - or writing an adhoc awk script (a very easy alternative, if you're familiar enough). Mark Robien On 3/1/2011 10:32 AM, Judith Reeks wrote: Dear All, I am currently refining a structure using the latest experimental version of Refmac (5.6) and there seems to be a problem with my Fo-Fc map. There is a region where I have
Re: [ccp4bb] I/sigmaI of 3.0 rule
First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
[ccp4bb] [Fwd: Re: [ccp4bb] I/sigmaI of 3.0 rule]
Original Message Subject:Re: [ccp4bb] I/sigmaI of 3.0 rule Date: Thu, 03 Mar 2011 10:43:23 -0700 From: Maia Cherney ch...@ualberta.ca To: Oganesyan, Vaheh oganesy...@medimmune.com References: 2ba9ce2f-c299-4ca9-a36a-99065d1b3...@unipd.it 4d6faed8.7040...@ualberta.ca 021001cbd9bc$f0ecc940$d2c65bc0$@gmail.com 4d6fcab6.3090...@ualberta.ca 4d6fcbff.2010...@ualberta.ca 73e543de77290c409c9bed6fa4ca34bb0173a...@md1ev002.medimmune.com Vaheh, The problem was with Rmerg. As you can see at I/sigma=2.84, the Rmerge (R-factor) was 143%. I am asking this question because B. Rupp wrote However, there is a simple relation between I/sigI and R-merge (provided no other indecency has been done to the data). It simply is (BMC) Rm=0.8/I/sigI. Maybe my data are indecent? This is the whole LP file. Maia ** XSCALE (VERSION December 6, 2007)28-Aug-2009 ** Author: Wolfgang Kabsch Copy licensed until (unlimited) to Canadian Light Source, Saskatoon, Canada. No redistribution. ** CONTROL CARDS ** MAXIMUM_NUMBER_OF_PROCESSORS=8 SPACE_GROUP_NUMBER=180 UNIT_CELL_CONSTANTS= 150.1 150.1 81.8 90.0 90.0 120.0 OUTPUT_FILE=XSCALE.HKL FRIEDEL'S_LAW=TRUE INPUT_FILE= XDS_ASCII.HKL XDS_ASCII INCLUDE_RESOLUTION_RANGE= 40 2.25 THE DATA COLLECTION STATISTICS REPORTED BELOW ASSUMES: SPACE_GROUP_NUMBER= 180 UNIT_CELL_CONSTANTS= 150.10 150.1081.80 90.000 90.000 120.000 * 12 EQUIVALENT POSITIONS IN SPACE GROUP #180 * If x',y',z' is an equivalent position to x,y,z, then x'=x*ML(1)+y*ML( 2)+z*ML( 3)+ML( 4)/12.0 y'=x*ML(5)+y*ML( 6)+z*ML( 7)+ML( 8)/12.0 z'=x*ML(9)+y*ML(10)+z*ML(11)+ML(12)/12.0 #1 2 3 45 6 7 89 10 11 12 11 0 0 00 1 0 00 0 1 0 20 -1 0 01 -1 0 00 0 1 8 3 -1 1 0 0 -1 0 0 00 0 1 4 4 -1 0 0 00 -1 0 00 0 1 0 50 1 0 0 -1 1 0 00 0 1 8 61 -1 0 01 0 0 00 0 1 4 70 1 0 01 0 0 00 0 -1 8 8 -1 0 0 0 -1 1 0 00 0 -1 4 91 -1 0 00 -1 0 00 0 -1 0 100 -1 0 0 -1 0 0 00 0 -1 8 111 0 0 01 -1 0 00 0 -1 4 12 -1 1 0 00 1 0 00 0 -1 0 ALL DATA SETS WILL BE SCALED TO XDS_ASCII.HKL ** READING INPUT REFLECTION DATA FILES ** DATAMEAN REFLECTIONSINPUT FILE NAME SET# INTENSITY ACCEPTED REJECTED 1 0.6203E+03 557303 0 XDS_ASCII.HKL ** CORRECTION FACTORS AS FUNCTION OF IMAGE NUMBER RESOLUTION ** RECIPROCAL CORRECTION FACTORS FOR INPUT DATA SETS MERGED TO OUTPUT FILE: XSCALE.HKL THE CALCULATIONS ASSUME FRIEDEL'S_LAW= TRUE TOTAL NUMBER OF CORRECTION FACTORS DEFINED 720 DEGREES OF FREEDOM OF CHI^2 FIT140494.9 CHI^2-VALUE OF FIT OF CORRECTION FACTORS 1.037 NUMBER OF CYCLES CARRIED OUT 3 CORRECTION FACTORS for visual inspection with VIEW DECAY_001.pck INPUT_FILE=XDS_ASCII.HKL XMIN= 0.1 XMAX= 179.9 NXBIN= 36 YMIN= 0.00257 YMAX= 0.19752 NYBIN= 20 NUMBER OF REFLECTIONS USED FOR DETERMINING CORRECTION FACTORS 238321 ** CORRECTION FACTORS AS FUNCTION OF X (fast) Y(slow) IN THE DETECTOR PLANE ** RECIPROCAL CORRECTION FACTORS FOR INPUT DATA SETS MERGED TO OUTPUT FILE: XSCALE.HKL THE CALCULATIONS ASSUME FRIEDEL'S_LAW= TRUE TOTAL NUMBER OF CORRECTION FACTORS DEFINED 4760 DEGREES OF FREEDOM OF CHI^2 FIT186486.8 CHI^2-VALUE OF FIT OF CORRECTION
Re: [ccp4bb] I/sigmaI of 3.0 rule
just to clarify that, at least in my case, my impression is that the editor was fair, I was referring only to the comment of one reviewer. Roberto Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it Il giorno 03/mar/2011, alle ore 18.16, Van Den Berg, Bert ha scritto: We should compile this discussion and send it as compulsive reading to journal editors...;-) Bert
Re: [ccp4bb] I/sigmaI of 3.0 rule
I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
Dear All, Relatively recent statistics on I/sigmaI and Rmerge in PDB deposits are presented in two following publications: 1.Benefits of structural genomics for drug discovery research. Grabowski M, Chruszcz M, Zimmerman MD, Kirillova O, Minor W. Infect Disord Drug Targets. 2009 Nov;9(5):459-74. PMID: 19594422 2. X-ray diffraction experiment-the last experiment in the structure elucidation process. Chruszcz M, Borek D, Domagalski M, Otwinowski Z, Minor W. Adv Protein Chem Struct Biol. 2009;77:23-40 PMID: 20663480 Best regards, Maksattachment: I_over_sigma_I.png
Re: [ccp4bb] I/sigmaI of 3.0 rule
Hello Maia, Rmerge is obsolete, so the reviewers had a good point to make you publish Rmeas instead. Rmeas should replace Rmerge in my opinion. The data statistics you sent show a mulltiplicity of about 20! Did you check your data for radiation damage? That might explain why your Rmeas is so utterly high while your I/sigI is still above 2 (You should not cut your data but include more!) What do the statistics look like if you process just about enough frames so that you get a reasonable mulltiplicity, 3-4, say? Cheers, Tim On Thu, Mar 03, 2011 at 10:57:37AM -0700, Maia Cherney wrote: I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it -- -- 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] I/sigmaI of 3.0 rule
The data statistics you sent show a mulltiplicity of about 20! Did you check your data for radiation damage? That might explain why your Rmeas is so utterly high while your I/sigI is still above 2 (You should not cut your data but include more!) So then I got that wrong - with that *high* a redundancy, the preceding term becomes ~1 and linear Rmerge and Rmeas asymptotically become the same? BR Cheers, Tim On Thu, Mar 03, 2011 at 10:57:37AM -0700, Maia Cherney wrote: I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
Re: [ccp4bb] I/sigmaI of 3.0 rule
Rmeas is always higher than Rmerge, so if my Rmerg is high I don't like Rmeas either. But that makes perfect sense now per Tim: the linear Rmerge gives for small N (lower redundancy) always lower values and rises with redundancy to approach Rmeas/rim for high redundancy. I like the idea just to look at the I/sigI and include more data. Lucky me to suggest to use all your present data for refinement... ;-) BR Maia Tim Gruene wrote: Hello Maia, Rmerge is obsolete, so the reviewers had a good point to make you publish Rmeas instead. Rmeas should replace Rmerge in my opinion. The data statistics you sent show a mulltiplicity of about 20! Did you check your data for radiation damage? That might explain why your Rmeas is so utterly high while your I/sigI is still above 2 (You should not cut your data but include more!) What do the statistics look like if you process just about enough frames so that you get a reasonable mulltiplicity, 3-4, say? Cheers, Tim On Thu, Mar 03, 2011 at 10:57:37AM -0700, Maia Cherney wrote: I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
[ccp4bb] setting up additive screen
Dear all, I am trying to optimize my crystal with additives. Since the yield of my protein purification is very limited, I am wondering what is the most efficient way to set up drops with additive to save my protein and not wasting additives? I am setting up 1 to 1 drops with 0.2 ul additives. But I feel 0.2ul is not very actuate, even if I use a p2. Would you share your ways to set up drops with additives? If I want to screen some additives, what additives would you suggest to try first, especially those from the 96 additive conditions from Hampton? By the way, just wondering, what kind of p2 pipettor work better? Any input is greatly appreciated! Thank you, Min
[ccp4bb] AKTA Explorer and Prime need new homes [off-topic]
Hello CCP4BB, We have a beautiful GE AKTA Explorer and an AKTA Prime available. Please contact me directly at ress...@gmail.com or 510-344-6633 for more info. Thanks, Erin
Re: [ccp4bb] I/sigmaI of 3.0 rule
I don't like Rmeas either. Given the Angst caused by actually useful redundancy, would it not be more reasonable then to report Rpim which decreases with redundancy? Maybe Rpim in an additional column would help to reduce the Angst? BR Maia Tim Gruene wrote: Hello Maia, Rmerge is obsolete, so the reviewers had a good point to make you publish Rmeas instead. Rmeas should replace Rmerge in my opinion. The data statistics you sent show a mulltiplicity of about 20! Did you check your data for radiation damage? That might explain why your Rmeas is so utterly high while your I/sigI is still above 2 (You should not cut your data but include more!) What do the statistics look like if you process just about enough frames so that you get a reasonable mulltiplicity, 3-4, say? Cheers, Tim On Thu, Mar 03, 2011 at 10:57:37AM -0700, Maia Cherney wrote: I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way below I/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of 3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] I/sigmaI of 3.0 rule
higher redundancy lowers Rpim because it increases precision. However, it need not increase accuracy if the observations are not drawn from the true distribution. If pathologic behaviour of Rfactor statistics is due to radiation damage, as I believe is often the case, we are combining observations that are no longer equivalent. If you used long exposures per image and collected just enough data for a complete data set you are out of luck. If you used shorter exposures and opted for a high-redundancy set then you have the option to toss out the last N images to get rid of the most damaged data, or you can try to compensate for the damage with zerodose, or whatever the name was of the program, I think from Wolfgang Kabsch. Rejecting data is never desirable but I think it may be better than merging non-equivalent data that can't be properly modeled by a single structure. Bart On 11-03-03 12:34 PM, Bernhard Rupp (Hofkristallrat a.D.) wrote: I don't like Rmeas either. Given the Angst caused by actually useful redundancy, would it not be more reasonable then to report Rpim which decreases with redundancy? Maybe Rpim in an additional column would help to reduce the Angst? BR Maia Tim Gruene wrote: Hello Maia, Rmerge is obsolete, so the reviewers had a good point to make you publish Rmeas instead. Rmeas should replace Rmerge in my opinion. The data statistics you sent show a mulltiplicity of about 20! Did you check your data for radiation damage? That might explain why your Rmeas is so utterly high while your I/sigI is still above 2 (You should not cut your data but include more!) What do the statistics look like if you process just about enough frames so that you get a reasonable mulltiplicity, 3-4, say? Cheers, Tim On Thu, Mar 03, 2011 at 10:57:37AM -0700, Maia Cherney wrote: I see, there is no consensus about my data. Some people say 2.4A, other say all. Well, I chose 2.3 A. My rule was to be a little bit below Rmerg 100%. At 2.3A Rmerg was 98.7% Actually, I have published my paper in JMB. Yes, reviewers did not like that and even made me give Rrim and Rpim etc. Maia Bernhard Rupp (Hofkristallrat a.D.) wrote: First of all I would ask a XDS expert for that because I don't know exactly what stats the XDS program reports (shame on me, ok) nor what the quality of your error model is, or what you want to use the data for (I guess refinement - see Eleanor's response for that, and use all data). There is one point I'd like to make re cutoff: If one gets greedy and collects too much noise in high resolution shells (like way belowI/sigI = 0.8 or so) the scaling/integration may suffer from an overabundance of nonsense data, and here I believe it makes sense to select a higher cutoff (like what exactly?) and reprocess the data. Maybe one of our data collection specialist should comment on that. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Maia Cherney Sent: Thursday, March 03, 2011 9:13 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I/sigmaI of3.0 rule I have to resend my statistics. Maia Cherney wrote: Dear Bernhard I am wondering where I should cut my data off. Here is the statistics from XDS processing. Maia On 11-03-03 04:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it -- 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] setting up additive screen
Use robot. You only need 0.1ul*96=9.6ul of protein solution. On Thu, Mar 3, 2011 at 7:59 PM, m zhang mzhang...@hotmail.com wrote: Dear all, I am trying to optimize my crystal with additives. Since the yield of my protein purification is very limited, I am wondering what is the most efficient way to set up drops with additive to save my protein and not wasting additives? I am setting up 1 to 1 drops with 0.2 ul additives. But I feel 0.2ul is not very actuate, even if I use a p2. Would you share your ways to set up drops with additives? If I want to screen some additives, what additives would you suggest to try first, especially those from the 96 additive conditions from Hampton? By the way, just wondering, what kind of p2 pipettor work better? Any input is greatly appreciated! Thank you, Min
[ccp4bb] mosflm gain
wondering if mosflm can automatically estimate the gain. i.e. i gather it is still estimated the usual way. -Bryan
Re: [ccp4bb] Processing Laue data
Based on roughly 1500 complete Laue data sets containing more than 45000 Laue patterns, I can say the following: How to collect Laue data? 1.) put crystal on (capillary or cryo is fine) 2.) switch on X-rays (or Neutrons) for time-resolved studies select one or more pulses else forget pulses 2a) for time-resolved studies scan the crystal edge at reduced Xray-flux to skim only surface that is hit by the Laser after edge scan expose to full X-ray pulses else forget about edge scan 3.) switch off X-rays (or Neutrons) 4.) read out pattern 5.) set crystal to another orientation delta phi is dependent on the bandwidth (approx. formula available in Ren et al., 1999) is usually 2-3 deg with 10% bandwidth eg 0.1 A bandwidth at 1 A mean wavelength provided by undulators 6.) goto step 2.) and repeat until reciprocal space covered 7.) reduce data with program. in my perception, the BEST and MOST USER FRIENDLY for X-ray Laue is: Precognition/Epinorm (RenzResearch) 20 data sets per day can be achieved!!! (I achieved once 56 data sets in 2 days). Get a faster computer or more processors increases speed. 8.) based on an idea by Anfinrud/Schotte step 5 can be made much more complicated with randomly filling gaps. Very nice feature, since it fills reciprocal space randomly, and if crystal dies you have at least random (but not complete) coverage of reciprocal space. You may also translate the crystal a bit to expose a fresh pristine crystal volume. 9.) If it works (mosaicity can still work against you), enjoy data that are as good as monochromatic, and they will give perfect maps. 10.) refine model with CNS or refmac or any other refinement program. Best Marius To all Laue experts up here How does a Laue data is collected? Thanks in advance to all PSP On Fri, Jan 28, 2011 at 3:43 AM, REX PALMER rex.pal...@btinternet.comwrote: What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College -- Pius S Padayatti Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
Re: [ccp4bb] Processing Laue data
Dear John, of course you are right, apologies for my little exaggeration. Warmest regards Marius Dear Marius, To these two centres to which you refer we can add:- Diamond Light Source; contact person Prof David Allan (small molecule Laue X-ray crystallography); Institut Laue Langevin ; contact person Dr Matthew Blakeley (neutron Laue protein crystallography); Los Alamos Neutron Source; contact person Dr Paul Langan (neutron time-of-flight Laue protein crystallography). Also, just a historical note; the SRS wiggler 9 Laue effort I deliberately recentred at ESRF ID09 when Michael Wulff joined ESRF and set about, with SAC and community approval, buidling ID09. Yours sincerely, John On Wed, Mar 2, 2011 at 12:14 AM, Marius Schmidt marius.schm...@ph.tum.de wrote: there is a small but brave community that actually attempted to collect Laue data on proteins with modern synchrotron sources. They are all centered around Keith Moffat in Chicago and Michael Wulff in Grenoble. Maybe you contact these people: D. Bourgeois at the ESRF, V. Srajer or Z. Ren at the APS. Best Marius What programs are available for processing Laue data to produce an intensity data set? Are explanatory notes or publications available? Rex Palmer Birkbeck College Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/ -- Professor John R Helliwell DSc Dr.habil. Marius Schmidt Asst. Professor University of Wisconsin-Milwaukee Department of Physics Room 454 1900 E. Kenwood Blvd. Milwaukee, WI 53211 phone: +1-414-229-4338 email: m-schm...@uwm.edu http://users.physik.tu-muenchen.de/marius/
Re: [ccp4bb] mosflm gain
Usually Mosflm will use a default value for the gain that depends on the type of detector used. This value is not realistic for CCD detectors, that is it is not really equal to the ratio of ADUs to incident X-ray photons, however it satisfies typical images under the assumptions of pixel independence and Poisson distribution, which are not true either. Inasmuch as the gain is just a scale factor in the data, it doesn't really matter that it isn't physically meaningful in the way you might expect from its name. However, the procedure of calculating gain from the variance to mean ratio from a background region of the image, which is the only simple automatic approach available if all you have is an image, should be avoided if you are looking for the gain in real units. I realise that didn't answer the question, but I thought it might be worth pointing out. -- David On 3 March 2011 20:34, Bryan Lepore bryanlep...@gmail.com wrote: wondering if mosflm can automatically estimate the gain. i.e. i gather it is still estimated the usual way. -Bryan
Re: [ccp4bb] I/sigmaI of 3.0 rule
not sure whether this option has been mentioned before ... i think what we really would like to do is decide by the quality of the density. i see that this is difficult. so, short of that ... how about the figure of merit in refinement ? wouldn't the fom reflect how useful our data really are ? ingo On 03/03/2011 12:29, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
