Re: [ccp4bb] packing test PHASER
I agree that looking at the packing is a good idea, but I also agree that having the wrong space group is a likely possible explanation. That’s the most common scenario when there are many clashing solutions with high TFZ scores. Randy Read On 17 Jun 2014, at 15:52, Roger Rowlett wrote: > Increase the number of allowed clashes in Phaser, re-run it then look at the > packing of the solution found and identify the source of the clashes. > Possibilities for the clash issue include: > Wrong space group > Flexible loops or termini in search model not present or differently > arranged in your crystal target > Once you look at the packing in Coot or Pymol, you will have a good idea of > what to do next. If the problem is flexible loops or misplaced N- or > C-termini, you can delete these regions from your search model (they are not > helping you phase anyway) and re-run Phaser with an appropriately truncated > search model. > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > On 6/17/2014 10:44 AM, Almudena Ponce Salvatierra wrote: >> Dear ccp4 users, >> >> I get the following message from Phaser when I do a molecular replacement >> with two ensembles. One of the ensembles is placed but the second one is not >> placed, and then it says this: >> >> Solutions with Z-scores greater than 13.0 (the threshold indicating a >> definite >> solution) were rejected for failing packing test >> #1 TFZ=16.8 PAK=487 >> #2 TFZ=17.4 PAK=440 >> #3 TFZ=17.0 PAK=312 >> #4 TFZ=16.7 PAK=294 >> #5 TFZ=16.8 PAK=227 >> #6 TFZ=16.6 PAK=284 >> #7 TFZ=16.2 PAK=219 >> #8 TFZ=16.3 PAK=287 >> #9 TFZ=16.1 PAK=186 >> #10 TFZ=16.6 PAK=277 >> #11 TFZ=16.7 PAK=204 >> #12 TFZ=16.8 PAK=404 >> #13 TFZ=15.9 PAK=271 >> #14 TFZ=15.8 PAK=194 >> #15 TFZ=14.9 PAK=229 >> #16 TFZ=16.4 PAK=368 >> #17 TFZ=17.3 PAK=194 >> #18 TFZ=15.2 PAK=240 >> #19 TFZ=16.1 PAK=325 >> #20 TFZ=15.3 PAK=455 >> #21 TFZ=16.5 PAK=298 >> #22 TFZ=16.6 PAK=290 >> #23 TFZ=15.2 PAK=259 >> #24 TFZ=16.2 PAK=194 >> #25 TFZ=16.1 PAK=314 >> #26 TFZ=16.3 PAK=194 >> #27 TFZ=16.3 PAK=387 >> #28 TFZ=15.6 PAK=193 >> #29 TFZ=15.7 PAK=219 >> #30 TFZ=15.6 PAK=474 >> #31 TFZ=15.1 PAK=194 >> #32 TFZ=15.4 PAK=339 >> #33 TFZ=15.5 PAK=210 >> #34 TFZ=15.2 PAK=300 >> #35 TFZ=15.6 PAK=186 >> #36 TFZ=16.2 PAK=216 >> #37 TFZ=14.9 PAK=182 >> #38 TFZ=15.7 PAK=279 >> #39 TFZ=15.5 PAK=285 >> #40 TFZ=15.0 PAK=374 >> #41 TFZ=15.4 PAK=404 >> #42 TFZ=15.3 PAK=185 >> #43 TFZ=15.6 PAK=227 >> #44 TFZ=14.8 PAK=364 >> #45 TFZ=15.7 PAK=193 >> #46 TFZ=14.5 PAK=448 >> #47 TFZ=14.6 PAK=219 >> #48 TFZ=15.5 PAK=210 >> #49 TFZ=15.7 PAK=189 >> #50 TFZ=15.0 PAK=193 >> #51 TFZ=15.0 PAK=281 >> #52 TFZ=15.2 PAK=202 >> >> And the list still continues for a bit. How should I think about this? I >> would assume that the clashes are too many only by seeing those numbers, but >> maybe there is something else to take into account? >> >> Thanks a lot in advance. >> >> Best wishes, >> >> Almudena >> >> >> -- >> Almudena Ponce-Salvatierra >> Macromolecular crystallography and Nucleic acid chemistry >> Max Planck Institute for Biophysical Chemistry >> Am Fassberg 11 37077 Göttingen >> Germany >> > -- 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] packing test PHASER
Roger, Given that PAK value is the number of clashing residues I doubt that in this case it is a loop clash etc. As a general comment though you are absolutely correct. Cheers, Ed Sent on a Sprint Samsung Galaxy S® III Original message From: Roger Rowlett Date:06/17/2014 10:52 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] packing test PHASER Increase the number of allowed clashes in Phaser, re-run it then look at the packing of the solution found and identify the source of the clashes. Possibilities for the clash issue include: Wrong space group Flexible loops or termini in search model not present or differently arranged in your crystal target Once you look at the packing in Coot or Pymol, you will have a good idea of what to do next. If the problem is flexible loops or misplaced N- or C-termini, you can delete these regions from your search model (they are not helping you phase anyway) and re-run Phaser with an appropriately truncated search model. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/17/2014 10:44 AM, Almudena Ponce Salvatierra wrote: Dear ccp4 users, I get the following message from Phaser when I do a molecular replacement with two ensembles. One of the ensembles is placed but the second one is not placed, and then it says this: Solutions with Z-scores greater than 13.0 (the threshold indicating a definite solution) were rejected for failing packing test #1 TFZ=16.8 PAK=487 #2 TFZ=17.4 PAK=440 #3 TFZ=17.0 PAK=312 #4 TFZ=16.7 PAK=294 #5 TFZ=16.8 PAK=227 #6 TFZ=16.6 PAK=284 #7 TFZ=16.2 PAK=219 #8 TFZ=16.3 PAK=287 #9 TFZ=16.1 PAK=186 #10 TFZ=16.6 PAK=277 #11 TFZ=16.7 PAK=204 #12 TFZ=16.8 PAK=404 #13 TFZ=15.9 PAK=271 #14 TFZ=15.8 PAK=194 #15 TFZ=14.9 PAK=229 #16 TFZ=16.4 PAK=368 #17 TFZ=17.3 PAK=194 #18 TFZ=15.2 PAK=240 #19 TFZ=16.1 PAK=325 #20 TFZ=15.3 PAK=455 #21 TFZ=16.5 PAK=298 #22 TFZ=16.6 PAK=290 #23 TFZ=15.2 PAK=259 #24 TFZ=16.2 PAK=194 #25 TFZ=16.1 PAK=314 #26 TFZ=16.3 PAK=194 #27 TFZ=16.3 PAK=387 #28 TFZ=15.6 PAK=193 #29 TFZ=15.7 PAK=219 #30 TFZ=15.6 PAK=474 #31 TFZ=15.1 PAK=194 #32 TFZ=15.4 PAK=339 #33 TFZ=15.5 PAK=210 #34 TFZ=15.2 PAK=300 #35 TFZ=15.6 PAK=186 #36 TFZ=16.2 PAK=216 #37 TFZ=14.9 PAK=182 #38 TFZ=15.7 PAK=279 #39 TFZ=15.5 PAK=285 #40 TFZ=15.0 PAK=374 #41 TFZ=15.4 PAK=404 #42 TFZ=15.3 PAK=185 #43 TFZ=15.6 PAK=227 #44 TFZ=14.8 PAK=364 #45 TFZ=15.7 PAK=193 #46 TFZ=14.5 PAK=448 #47 TFZ=14.6 PAK=219 #48 TFZ=15.5 PAK=210 #49 TFZ=15.7 PAK=189 #50 TFZ=15.0 PAK=193 #51 TFZ=15.0 PAK=281 #52 TFZ=15.2 PAK=202 And the list still continues for a bit. How should I think about this? I would assume that the clashes are too many only by seeing those numbers, but maybe there is something else to take into account? Thanks a lot in advance. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] Post doctoral position at Institute of Biotechnology, Univ. Helsinki
A postdoctoral position in structural biology is available from Sept 2014 earliest in the research group lead by Tommi Kajander at Institute of Biotechnology, University of Helsinki, Finland. The position is funded by the Academy of Finland at the Finnish university salary scale 5. The goal of the project is to understand the molecular basis of mammalian adhesion protein functions and solve structures of relevant protein-protein complexes. We are utilizing X-ray crystallography as the main methods to solve protein structures, with complementary methods such as SAXS, NMR and single particle EM and protein engineering and functional in vitro and cell-based studies. The topics of research projects will be discussed in detail during the interview of the selected candidates. Host institute has excellent infrastructure for structural biology studies. Applicants with strong background in protein expression and purification and molecular biology, and experience in crystallography/structural biology techniques are encouraged to apply (in particular experience with insect and eukaryotic expression systems is a plus). The applicant should be a team player with good English and communication skills and capability to guide studends. Applicants should send 1) a cover letter briefly describing previous achievements, future career ambitions and motivation for this position; 2) Curriculum vitae with a list of publications and contact information of two referees should be sent via email to: tommi.kajander-at-helsinki.fi The deadline for applications is July 31st, 2014, or until suitable candidates are found. Please feel free to contact for more information. Tommi Kajander, Ph.D. Team Leader Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-50-4480991 tommi . kajander-at-helsinki . fi http://www.biocenter.helsinki.fi/bi/kajander/
Re: [ccp4bb] packing test PHASER
Increase the number of allowed clashes in Phaser, re-run it then look at the packing of the solution found and identify the source of the clashes. Possibilities for the clash issue include: 1. Wrong space group 2. Flexible loops or termini in search model not present or differently arranged in your crystal target Once you look at the packing in Coot or Pymol, you will have a good idea of what to do next. If the problem is flexible loops or misplaced N- or C-termini, you can delete these regions from your search model (they are not helping you phase anyway) and re-run Phaser with an appropriately truncated search model. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 6/17/2014 10:44 AM, Almudena Ponce Salvatierra wrote: Dear ccp4 users, I get the following message from Phaser when I do a molecular replacement with two ensembles. One of the ensembles is placed but the second one is not placed, and then it says this: Solutions with Z-scores greater than 13.0 (the threshold indicating a definite solution) were rejected for failing packing test #1 TFZ=16.8 PAK=487 #2 TFZ=17.4 PAK=440 #3 TFZ=17.0 PAK=312 #4 TFZ=16.7 PAK=294 #5 TFZ=16.8 PAK=227 #6 TFZ=16.6 PAK=284 #7 TFZ=16.2 PAK=219 #8 TFZ=16.3 PAK=287 #9 TFZ=16.1 PAK=186 #10 TFZ=16.6 PAK=277 #11 TFZ=16.7 PAK=204 #12 TFZ=16.8 PAK=404 #13 TFZ=15.9 PAK=271 #14 TFZ=15.8 PAK=194 #15 TFZ=14.9 PAK=229 #16 TFZ=16.4 PAK=368 #17 TFZ=17.3 PAK=194 #18 TFZ=15.2 PAK=240 #19 TFZ=16.1 PAK=325 #20 TFZ=15.3 PAK=455 #21 TFZ=16.5 PAK=298 #22 TFZ=16.6 PAK=290 #23 TFZ=15.2 PAK=259 #24 TFZ=16.2 PAK=194 #25 TFZ=16.1 PAK=314 #26 TFZ=16.3 PAK=194 #27 TFZ=16.3 PAK=387 #28 TFZ=15.6 PAK=193 #29 TFZ=15.7 PAK=219 #30 TFZ=15.6 PAK=474 #31 TFZ=15.1 PAK=194 #32 TFZ=15.4 PAK=339 #33 TFZ=15.5 PAK=210 #34 TFZ=15.2 PAK=300 #35 TFZ=15.6 PAK=186 #36 TFZ=16.2 PAK=216 #37 TFZ=14.9 PAK=182 #38 TFZ=15.7 PAK=279 #39 TFZ=15.5 PAK=285 #40 TFZ=15.0 PAK=374 #41 TFZ=15.4 PAK=404 #42 TFZ=15.3 PAK=185 #43 TFZ=15.6 PAK=227 #44 TFZ=14.8 PAK=364 #45 TFZ=15.7 PAK=193 #46 TFZ=14.5 PAK=448 #47 TFZ=14.6 PAK=219 #48 TFZ=15.5 PAK=210 #49 TFZ=15.7 PAK=189 #50 TFZ=15.0 PAK=193 #51 TFZ=15.0 PAK=281 #52 TFZ=15.2 PAK=202 And the list still continues for a bit. How should I think about this? I would assume that the clashes are too many only by seeing those numbers, but maybe there is something else to take into account? Thanks a lot in advance. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] packing test PHASER
Dear ccp4 users, I get the following message from Phaser when I do a molecular replacement with two ensembles. One of the ensembles is placed but the second one is not placed, and then it says this: Solutions with Z-scores greater than 13.0 (the threshold indicating a definite solution) were rejected for failing packing test #1 TFZ=16.8 PAK=487 #2 TFZ=17.4 PAK=440 #3 TFZ=17.0 PAK=312 #4 TFZ=16.7 PAK=294 #5 TFZ=16.8 PAK=227 #6 TFZ=16.6 PAK=284 #7 TFZ=16.2 PAK=219 #8 TFZ=16.3 PAK=287 #9 TFZ=16.1 PAK=186 #10 TFZ=16.6 PAK=277 #11 TFZ=16.7 PAK=204 #12 TFZ=16.8 PAK=404 #13 TFZ=15.9 PAK=271 #14 TFZ=15.8 PAK=194 #15 TFZ=14.9 PAK=229 #16 TFZ=16.4 PAK=368 #17 TFZ=17.3 PAK=194 #18 TFZ=15.2 PAK=240 #19 TFZ=16.1 PAK=325 #20 TFZ=15.3 PAK=455 #21 TFZ=16.5 PAK=298 #22 TFZ=16.6 PAK=290 #23 TFZ=15.2 PAK=259 #24 TFZ=16.2 PAK=194 #25 TFZ=16.1 PAK=314 #26 TFZ=16.3 PAK=194 #27 TFZ=16.3 PAK=387 #28 TFZ=15.6 PAK=193 #29 TFZ=15.7 PAK=219 #30 TFZ=15.6 PAK=474 #31 TFZ=15.1 PAK=194 #32 TFZ=15.4 PAK=339 #33 TFZ=15.5 PAK=210 #34 TFZ=15.2 PAK=300 #35 TFZ=15.6 PAK=186 #36 TFZ=16.2 PAK=216 #37 TFZ=14.9 PAK=182 #38 TFZ=15.7 PAK=279 #39 TFZ=15.5 PAK=285 #40 TFZ=15.0 PAK=374 #41 TFZ=15.4 PAK=404 #42 TFZ=15.3 PAK=185 #43 TFZ=15.6 PAK=227 #44 TFZ=14.8 PAK=364 #45 TFZ=15.7 PAK=193 #46 TFZ=14.5 PAK=448 #47 TFZ=14.6 PAK=219 #48 TFZ=15.5 PAK=210 #49 TFZ=15.7 PAK=189 #50 TFZ=15.0 PAK=193 #51 TFZ=15.0 PAK=281 #52 TFZ=15.2 PAK=202 And the list still continues for a bit. How should I think about this? I would assume that the clashes are too many only by seeing those numbers, but maybe there is something else to take into account? Thanks a lot in advance. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] Hosed-Up X-Ray Structures: A Big Problem
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Pavel, maybe I should have explained in better detail to avoid confusion. The importance of the contribution to the X-ray term from hydrogens has been well-known (and used in refinement programs) for - I guess - more than 40 years, but I am sure you know that. I meant to say that small variations in the hydrogen position don't show up for X-ray data, which is why they can be calculated even at 0.8A resolution. Maybe you could repeat the study you point at and move the hydrogen atoms, e.g. 0.3A out of their calculated position - my guess is such a variation would no show up in the map or the R-values at resolutions ranges significant for macromolecules. But these differences may matter when it comes to quantum chemical calculations, and Jeffrey's post seems to support this. I am puzzled that you observe the same with neutron data, i.e. you see no difference between the riding hydrogen model and restraining them. Does phenix, by any chance, refine the B-values rather than constrain them as implied by 'riding atom model'? This would be very unusual and might explain why the differences are not detected with phenix. In our work using shelxl, which I mentioned before, the quality differences where really striking despite the low data completeness. Interesting that phenix seems to wipe out the most interesting part of (MX) neutron data. Cheers, Tim On 06/16/2014 08:09 PM, Pavel Afonine wrote: > Hi Tim, > > just to spice your words up with some numbers > > You may also want to note that constrained hydrogen positions are > a >> crude approximation and only work with X-ray data where hydrogen >> atoms have little impact on the data. > > > This contribution can be as large as 1.5% difference in R-factor > (with vs without H), as shown in Figure 2 (page 19; "On the > contribution of hydrogen atoms to X-ray scattering"): > http://phenix-online.org/newsletter/CCN_2012_01.pdf > > >> Our comparison between hydrogen restraints and constraints >> (http://dx.doi.org/10.1107/S1600576713027659) report the greater >> quality of restraints vs. constraints when it comes to neutron >> data, where hydrogen atoms do matter. > > > I just re-refined (phenix.refine) all neutron structures available > in PDB (for which I could extract diffraction data without manual > labor; 55 in total) with two ways of handling H (D and H/D) atoms: > a) refine H individually, and b) using riding model for H > (rotatable H are adjusted to fit the map). In terms of Rfree and > Rwork I don't see a huge difference. However, using riding model > results in less overfitting: > http://cci.lbl.gov/~afonine/tmp/r_stats.pdf > > This is not surprising given typical quality of neutron data: > average (all neutron entries in PDB) completeness of neutron data > sets is 76%, while average completeness of comparable X-ray data > sets is 94% (page 21): > http://phenix-online.org/presentations/latest/2012_afonine_ecm27-final.pdf > > All the best, Pavel > - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFToEdAUxlJ7aRr7hoRAk/2AJ9WEomb6EVbAj7vkhHq3cv6l1YivQCcCGgc 4ydPJJRvRwer6SnDnvhvG+k= =rG3w -END PGP SIGNATURE-
