Re: [ccp4bb] Phaser question, twinning, DIALS, suggestions welcome
Hi Jurgen, You could send me a logfile off-list, and maybe I would spot something in there. We’ve put some effort into putting more intelligence into the Phaser search, so that it adapts to the initial perceived difficulty of the problem in setting the initial parameters, and then adapts to indicators of success or failure during the search. Much of the time this works very well, but there’s obviously room for improvement. For one thing, it appears that we’re frequently too optimistic about how good the model will be and how easy the search will be. One question: when you say that you cut at 2.5A for MR, do you do that by setting the resolution within the Phaser run, or do you have an MTZ file with only data to 2.5A? If the former, then you’re over-riding some of Phaser’s automation, which will choose the initial resolution limit based on the perceived difficulty of the problem (a function of model completeness, expected RMS error of the model, and the number of reflections to different resolution limits). It’s this initial automated choice that can go wrong if we’re too optimistic, because then a clear solution isn’t found, and then Phaser repeats the search with data to the full resolution, which can take longer than just choosing an intermediate resolution from the start. Anyway, if the problem is expected to be easy but turns out to be difficult, this implies that some of the information used to decide it should be easy is wrong or too optimistic. One top possibility is that the model is not as good as expected, e.g. because of conformational changes. If there’s a potential hinge-bending motion, then you’re usually better off searching with separate domains. If the change is something that can’t be described with rigid-body motions, then it would be better to increase the expected RMS error from what Phaser deduces from the sequence identity. Increasing the RMSD by 10-20% would be a good first bet in such a case. The other top possibility is that the space group is wrong. Is there any ambiguity in the space group? In particular, do any of the twinning tests indicate that the data may be twinned (which can lead to choosing too high symmetry)? Best wishes, Randy On 1 Oct 2014, at 03:02, Jurgen Bosch jbos...@jhu.edu wrote: Dear BB, or in particular Phaser developers :-) This must be part of British humor right (or was that the Canadian influence Randy) ? eLLG indicates that placement of ensemble ensemble_1 will be straightforward The data are sufficient to exceed the eLLG target The search space is finite 143 x 143 x 80 Å with 3 or 4 molecules per asu, but Phaser has been burning CPU cycles quite a bit, we are approaching 24h by now. Data extends to 1.7 Å - for MR we cut at 2.5 Å. I can hear Garib, yes, Molrep was done in few minutes but I’m not super convinced about the solution either. Same with BALBES and MrBump (which took a few more minutes, actually also days for MrBump) The space group appears to be P63 22 as judged by XDS, pointless, xtriage, however the crystals are split (at least in some areas it’s visible) but I thought XDS would take care of these “aliens” and eliminate them mostly. My graduate student, Lauren, found a nifty program called DIALS that we wish to explore further to rescue the “nice” data we have and hopefully solve the structure. Lower symmetry space groups were tried down to P21 with increasing number of molecules per asu and applying twin laws if necessary. The minor problem with the twins is how do I really know that it is a higher symmetry space group and not a 50% twin in a lower symmetry ? Rwork/Rfree in this particular case do not seem to help at all for distinguishing between the solutions. Maps looks sort of right but R-factors are not reflecting what you see on the screen. We appreciate any suggestions and ideas what else we could do. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Phaser question, twinning, DIALS, suggestions welcome
Hi again, I should have mentioned that, if you have a good enough model, it’s often possible to solve the structure in P1. The molecular replacement solution will settle on one of the twin domains (or you may end up with more than one solution, related by the twin law(s)). Then the symmetry of the solution will tell you the true symmetry of the crystal. Otherwise, you’ll have to test all the subgroups, and there will be a lot of those for P6322, especially if you’re not sure about screw axes. But the true point group symmetry will only be lower than the symmetry in which the data merge if there’s twinning, which is why I’m wondering whether there are indications of twinning. It probably bears repeating that, if you test data merged in too low symmetry, the twinning tests that depend on twin laws can be misleading, because they look for reflections that are similar over a possible twin law. You need to have independent evidence from tests that do not depend on twin laws (i.e. statistical tests such as the moment tests or the L-test) that the data are perturbed in a way that implies twinning. Randy On 1 Oct 2014, at 03:02, Jurgen Bosch jbos...@jhu.edu wrote: Dear BB, or in particular Phaser developers :-) This must be part of British humor right (or was that the Canadian influence Randy) ? eLLG indicates that placement of ensemble ensemble_1 will be straightforward The data are sufficient to exceed the eLLG target The search space is finite 143 x 143 x 80 Å with 3 or 4 molecules per asu, but Phaser has been burning CPU cycles quite a bit, we are approaching 24h by now. Data extends to 1.7 Å - for MR we cut at 2.5 Å. I can hear Garib, yes, Molrep was done in few minutes but I’m not super convinced about the solution either. Same with BALBES and MrBump (which took a few more minutes, actually also days for MrBump) The space group appears to be P63 22 as judged by XDS, pointless, xtriage, however the crystals are split (at least in some areas it’s visible) but I thought XDS would take care of these “aliens” and eliminate them mostly. My graduate student, Lauren, found a nifty program called DIALS that we wish to explore further to rescue the “nice” data we have and hopefully solve the structure. Lower symmetry space groups were tried down to P21 with increasing number of molecules per asu and applying twin laws if necessary. The minor problem with the twins is how do I really know that it is a higher symmetry space group and not a 50% twin in a lower symmetry ? Rwork/Rfree in this particular case do not seem to help at all for distinguishing between the solutions. Maps looks sort of right but R-factors are not reflecting what you see on the screen. We appreciate any suggestions and ideas what else we could do. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Phaser question, twinning, DIALS, suggestions welcome
Dear Jurgen, Thanks for your interest in DIALS - we are working hard at the moment on testing the software and finding bugs (and fixing them!) and I would say right now it's not quite ready for the general user, but we do plan to make an alpha release of the software before the end of the year. When we're ready, we'll send an email out to the CCP4 BB of course. In the meantime some more information may be found at: http://dials.sourceforge.net/ Best wishes Graeme, and the DIALS development team On 1 October 2014 03:02, Jurgen Bosch jbos...@jhu.edu wrote: Dear BB, or in particular Phaser developers :-) This must be part of British humor right (or was that the Canadian influence Randy) ? eLLG indicates that placement of ensemble ensemble_1 will be straightforward The data are sufficient to exceed the eLLG target The search space is finite 143 x 143 x 80 Å with 3 or 4 molecules per asu, but Phaser has been burning CPU cycles quite a bit, we are approaching 24h by now. Data extends to 1.7 Å - for MR we cut at 2.5 Å. I can hear Garib, yes, Molrep was done in few minutes but I’m not super convinced about the solution either. Same with BALBES and MrBump (which took a few more minutes, actually also days for MrBump) The space group appears to be P63 22 as judged by XDS, pointless, xtriage, however the crystals are split (at least in some areas it’s visible) but I thought XDS would take care of these “aliens” and eliminate them mostly. My graduate student, Lauren, found a nifty program called DIALS that we wish to explore further to rescue the “nice” data we have and hopefully solve the structure. Lower symmetry space groups were tried down to P21 with increasing number of molecules per asu and applying twin laws if necessary. The minor problem with the twins is how do I really know that it is a higher symmetry space group and not a 50% twin in a lower symmetry ? Rwork/Rfree in this particular case do not seem to help at all for distinguishing between the solutions. Maps looks sort of right but R-factors are not reflecting what you see on the screen. We appreciate any suggestions and ideas what else we could do. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Phaser question, twinning, DIALS, suggestions welcome
Thanks Randy, so from your reply it seems that cutoff is differently treated. And if I interpret your email correctly it is better to provide Phaser with a truncated versus a full data set. I tried both cases, but I had assumed that if you restrict the resolution within Phaser it would be the same as if you give Phaser to begin with a truncated dataset. Perhaps it would be a good idea in th automatic routine to first check if the user wants to truncate the data and then run the automatic scoring analysis with that value ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Oct 1, 2014, at 5:15 AM, Randy Read rj...@cam.ac.ukmailto:rj...@cam.ac.uk wrote: Hi Jurgen, You could send me a logfile off-list, and maybe I would spot something in there. We’ve put some effort into putting more intelligence into the Phaser search, so that it adapts to the initial perceived difficulty of the problem in setting the initial parameters, and then adapts to indicators of success or failure during the search. Much of the time this works very well, but there’s obviously room for improvement. For one thing, it appears that we’re frequently too optimistic about how good the model will be and how easy the search will be. One question: when you say that you cut at 2.5A for MR, do you do that by setting the resolution within the Phaser run, or do you have an MTZ file with only data to 2.5A? If the former, then you’re over-riding some of Phaser’s automation, which will choose the initial resolution limit based on the perceived difficulty of the problem (a function of model completeness, expected RMS error of the model, and the number of reflections to different resolution limits). It’s this initial automated choice that can go wrong if we’re too optimistic, because then a clear solution isn’t found, and then Phaser repeats the search with data to the full resolution, which can take longer than just choosing an intermediate resolution from the start. Anyway, if the problem is expected to be easy but turns out to be difficult, this implies that some of the information used to decide it should be easy is wrong or too optimistic. One top possibility is that the model is not as good as expected, e.g. because of conformational changes. If there’s a potential hinge-bending motion, then you’re usually better off searching with separate domains. If the change is something that can’t be described with rigid-body motions, then it would be better to increase the expected RMS error from what Phaser deduces from the sequence identity. Increasing the RMSD by 10-20% would be a good first bet in such a case. The other top possibility is that the space group is wrong. Is there any ambiguity in the space group? In particular, do any of the twinning tests indicate that the data may be twinned (which can lead to choosing too high symmetry)? Best wishes, Randy On 1 Oct 2014, at 03:02, Jurgen Bosch jbos...@jhu.edumailto:jbos...@jhu.edu wrote: Dear BB, or in particular Phaser developers :-) This must be part of British humor right (or was that the Canadian influence Randy) ? eLLG indicates that placement of ensemble ensemble_1 will be straightforward The data are sufficient to exceed the eLLG target The search space is finite 143 x 143 x 80 Å with 3 or 4 molecules per asu, but Phaser has been burning CPU cycles quite a bit, we are approaching 24h by now. Data extends to 1.7 Å - for MR we cut at 2.5 Å. I can hear Garib, yes, Molrep was done in few minutes but I’m not super convinced about the solution either. Same with BALBES and MrBump (which took a few more minutes, actually also days for MrBump) The space group appears to be P63 22 as judged by XDS, pointless, xtriage, however the crystals are split (at least in some areas it’s visible) but I thought XDS would take care of these “aliens” and eliminate them mostly. My graduate student, Lauren, found a nifty program called DIALS that we wish to explore further to rescue the “nice” data we have and hopefully solve the structure. Lower symmetry space groups were tried down to P21 with increasing number of molecules per asu and applying twin laws if necessary. The minor problem with the twins is how do I really know that it is a higher symmetry space group and not a 50% twin in a lower symmetry ? Rwork/Rfree in this particular case do not seem to help at all for distinguishing between the solutions. Maps looks sort of right but R-factors are not reflecting what you see on the screen. We appreciate any suggestions and ideas what else we could do. Thanks, Jürgen
[ccp4bb] Phaser question, twinning, DIALS, suggestions welcome
Dear BB, or in particular Phaser developers :-) This must be part of British humor right (or was that the Canadian influence Randy) ? eLLG indicates that placement of ensemble ensemble_1 will be straightforward The data are sufficient to exceed the eLLG target The search space is finite 143 x 143 x 80 Å with 3 or 4 molecules per asu, but Phaser has been burning CPU cycles quite a bit, we are approaching 24h by now. Data extends to 1.7 Å - for MR we cut at 2.5 Å. I can hear Garib, yes, Molrep was done in few minutes but I’m not super convinced about the solution either. Same with BALBES and MrBump (which took a few more minutes, actually also days for MrBump) The space group appears to be P63 22 as judged by XDS, pointless, xtriage, however the crystals are split (at least in some areas it’s visible) but I thought XDS would take care of these “aliens” and eliminate them mostly. My graduate student, Lauren, found a nifty program called DIALS that we wish to explore further to rescue the “nice” data we have and hopefully solve the structure. Lower symmetry space groups were tried down to P21 with increasing number of molecules per asu and applying twin laws if necessary. The minor problem with the twins is how do I really know that it is a higher symmetry space group and not a 50% twin in a lower symmetry ? Rwork/Rfree in this particular case do not seem to help at all for distinguishing between the solutions. Maps looks sort of right but R-factors are not reflecting what you see on the screen. We appreciate any suggestions and ideas what else we could do. Thanks, Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Phaser question
Just to follow up on this: The enormous increase between the TFZ= and TFZ== numbers is surprising, so I focused on that in my original answer to Fred's question. However, if anyone wants to know the difference between these, it's documented on this web page: http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement#Annotation. Best wishes, Randy Read On 5 Dec 2013, at 08:36, vellieux frederic.velli...@ibs.fr wrote: Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494) -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] Phaser question
Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494)
Re: [ccp4bb] Phaser question
Hi Fred, Send me the logfiles (off-line), because this shouldn't be happening and I'd like to have a look. That said, we've been seeing some similar problems in certain circumstances, i.e. B-factor refinement refines to significant negative B-factor values, and data at high resolution have very low signal-to-noise. If those are the circumstances, we're currently working on a fix and maybe we can get you to test it on your data once it's implemented. All the best, Randy On 5 Dec 2013, at 08:36, vellieux frederic.velli...@ibs.fr wrote: Hiyya all, I have a question about the latest Phaser output, concerning TFZ = and TFZ == . I do not know how to interpret outputs of the type TFZ = 5.2 TFZ == 54.1; TFZ = 5.8 TFZ == 63.0; TFZ = 6.4 TFZ == 19.7 (these are real TFZ figures coming from Phaser log files). I used to analyse the Phaser output using TFZ, when only a single number was given. Now with two figures to consider, I do not know what to think of it any more. Any ideas out there ? And best wishes for an enjoyable end-of-the-year season (be it in the cold or in the sun depending on which side of the planet you sit). Fred. -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494) -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] Phaser question
Dear all, In the phaser .sol file what do the two LLG's correspond to on the SOLU SET line eg SOLU SET RFZ=20.7 TFZ=35.4 PAK=0 LLG=1699 LLG=2821 Do they show an initial and a refined LLG or do they correspond to the rotation and translation function as in the Z scores? I checked the appropriate web page and didn't see anything immediate. http://www-structmed.cimr.cam.ac.uk/phaser/documentation/phaser-2.1_key.html#MR_solved_it Thanks, Simon
Re: [ccp4bb] Phaser question
I think the second LLG value is after refinement. But sometimes I see that the second LLG goes negative and I wonder what it means. For example: RFZ=3.1 TFZ=17.2 PAK=0 LLG=238 LLG=-603 -Konstantin On Wed, 30 Sep 2009, Simon Kolstoe wrote: Dear all, In the phaser .sol file what do the two LLG's correspond to on the SOLU SET line eg SOLU SET RFZ=20.7 TFZ=35.4 PAK=0 LLG=1699 LLG=2821 Do they show an initial and a refined LLG or do they correspond to the rotation and translation function as in the Z scores? I checked the appropriate web page and didn't see anything immediate. http://www-structmed.cimr.cam.ac.uk/phaser/documentation/phaser-2.1_key.html#MR_solved_it Thanks, Simon -- Konstantin Korotkov, Ph.D. Research Scientist University of Washington Department of Biochemistry Box 357742 Seattle, WA 98195-7742 (206)616-4512 k...@u.washington.edu --
Re: [ccp4bb] Phaser question
In a Phaser automated molecular replacement job, it does almost everything at a working resolution and then a final refinement at a final resolution. By default, the working resolution is 2.5A and the final resolution is the full resolution of the data set. So the second-last LLG is the one computed with the working data (probably to 2.5A) and the last one is the LLG computed with all the data. Both LLG values are after rigid-body refinement. If the final LLG goes negative, then this probably means that the model isn't as good as Phaser was assuming, and that this becomes a problem in particular for the data at higher resolution. Best wishes, Randy Read On 30 Sep 2009, at 16:50, Simon Kolstoe wrote: Dear all, In the phaser .sol file what do the two LLG's correspond to on the SOLU SET line eg SOLU SET RFZ=20.7 TFZ=35.4 PAK=0 LLG=1699 LLG=2821 Do they show an initial and a refined LLG or do they correspond to the rotation and translation function as in the Z scores? I checked the appropriate web page and didn't see anything immediate. http://www-structmed.cimr.cam.ac.uk/phaser/documentation/phaser-2.1_key.html#MR_solved_it Thanks, Simon -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www- structmed.cimr.cam.ac.uk
[ccp4bb] Phaser question !!!
Hi all !!! Is it possible to fix one domain and search for the second one in phaser !!! What are the keywords to use. I have a protein with two domains. It i give both the domains together or as two ensembles it is not finding the solution. If i give only one domain it is giving the solution (R and Rfee is 28 and 33). But it is not finding the second one. Thanks in advance for your suggestions.. regards John
