Re: [ccp4bb] pointless error-summary
Do note that all the Pointless 1.5.x versions had a serious bug and should not be used Phil On 19 Nov 2011, at 09:49, Rajesh kumar wrote: Thanks to Harry Powell, Phil Evans, De-Feng Li for suggestions and special thanks to Charles Ballard looking in to my data. The summary is Harry Powell and Phil Evans-Pointless needs update as newer version is up to version 1.6.8 - the latest copy is at ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.6.8.linux De-Feng Li- In the log file, Number of batches (1) show that the dataset used in Pointless have been scaled. In fact, the input dataset should be not scaled. You can try it again. Charles Ballard-I can reproduce the failure using pointless 1.4.10 (ccp4 6.1.13). It, however, runs successfully with pointless 1.5.22 (ccp4-6.2.0). 1.4.10 is throwing a lot of memory errors, so it is probably a memory problem. I would suggest using a more up-to-date pointless. You can get that either from CCP4, or just pointless itself from ftp://mrc-lmb.cam.ac.uk/pub/pre. ps - you will probably get more from the unmerged data Thanks Rajesh Date: Tue, 15 Nov 2011 09:17:34 -0500 From: ccp4...@hotmail.com Subject: [ccp4bb] pointless error To: CCP4BB@JISCMAIL.AC.UK Dear all, mtzdump shows my space group as 'C 2 2 21' (number 20) pointless gives error Space group is not in library. part of the log file is following. I appreciate all the help. Thanks Raj ** ** * POINTLESS * * 1.4.5* ** * Determine Laue group from unmerged intensities * * Phil Evans MRC LMB, Cambridge * * Uses cctbx routines by Ralf Grosse-Kunstleve et al.* ** ** Maximum resolution in file output_7-21.mtz:2.740 Columns for I, sigI: IMEAN SIGIMEAN Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: /opt/programs/ccp4/ccp4-6.1.13/lib/data/syminfo.lib === * Summary of test data read in: Resolution range accepted:47.922.74 Number of reflections = 18008 Number of observations = 18008 Number of parts= 18008 Number of batches = 1 Number of datasets = 1 Project: Crystal: unknown Dataset: unknown210711:15:24:58 Run number: 1 consists of batches 1 to 1 Average unit cell: 110.66 112.08 120.7890.0090.0090.00 Unit cell (from HKLIN file) used to derive lattice symmetry with tolerance 2.0 degrees 110.66 112.08 120.78 90.00 90.00 90.00 Tolerance (and delta) is the maximum deviation from the expected angle between two-fold axes in the lattice group Lattice point group: P 4 2 2 Reindexing or changing symmetry Reindex operator from input cell to lattice cell: [-1/2h-1/2k,-1/2h+1/2k,-l] h' = ( h k l ) (-0.5-0.5 0 ) (-0.5 0.5 0 ) ( 0 0 -1 ) Lattice unit cell after reindexing: deviation 0.73 degrees 78.75 78.75 120.78 90.00 90.00 90.73 Number of reflections = 10213 Number of observations = 18008 Average multiplicity = 1.8 Resolution range in list: 47.92 - 2.74 Intensity normalisation: B-factor = -13.3 Resolution range reset to47.92 to 3.00 using I/sigmaI cutoff6.0 4 pairs rejected for E^2 too large Overall CC for 6256 unrelated pairs: 0.014 N= 6256 Estimated expectation value of true correlation coefficient E(CC) = 0.973 Estimated sd(CC) = 1.110 / Sqrt(N) Number of reflections omitted from ice rings: 613 Estimated E(CC) of true correlation coefficient from identity = 0.973 Failed to find spacegroup in SYMINFO! Space group is not in library Symop: X,Y,Z Symop: Y,X,-Z Symop: -Y,-X,-Z Symop: -X,-Y,Z Space group is not in library !--SUMMARY_BEGIN-- BFONT COLOR=#FF8800 !--SUMMARY_END-- -- FATAL ERROR message: Space group is not in library -- !--SUMMARY_BEGIN-- /FONT/B !--SUMMARY_END--
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor On 11/20/2011 03:57 PM, Napoleão Valadares wrote: Thank you all for the replies. Felix Frolow, Dan Leahy, Hans Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot. I think I understand it now, I always thought the one ring to rule them all translated in the crystallography realms to one origin to rule them all. That probably means I have a long road in front of me. I'm still half confused, I definitely need to read more, as much as I read about symmetry and space groups I never seem to improve or get a better understanding, but I'll keep trying. About the same origin: The pdbs of both Solution-1 and Solution-2 present the same space group and cell, as observed opening the pdbs as text files or in pymol. When I open both maps on coot they are not superposed but present the same cell and origin. If I open both solutions on pymol they clash. If I generate the symmetry mates of both solutions none of them are superposed, instead they clash. But I think they are related as you all pointed, I'll check it out. Thank you all for your kind answers and your patience with a beginner. Regards from a sunny Brazil, Napo On 11/20/2011 2:58 AM, Felix Frolow wrote: Napoleao, It is so called alternative origins play a game with you. You do not change your structure by shifting 1/2 translation (or even combination of these translations) into directions of the main axes of your unit cell. Structure factors after this operation stay the same, however phases change systematically, producing however the same map features. Would I be a begin crystallographer now, I would read a bit more old fashioned books on crystallography such as probably Jensen and Stout… FF 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 mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote: Hello, I'm observing a very strange phenomena (at least to me, I'm a beginner). It is related to symmetry (I think). I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas 35% in the last shell) and a partially refined solution with R/Rfree 22/24, 166 aminoacids observed and around 30 solvent molecules. I'll call this Solution-1. The refinement was smooth, the densities were very clearly asking for the correct missing side chains and the map looks good. The space group I'm using is P212121, pointless and XDS agree with that (but me and pointless both have a long history of being wrong about space groups). Phenix.xtriage says there's no twinning. I took Solution-1 and used it as a template in a molecular replacement in the same space group (P212121) using the same mtz as the one used to refine the template. I got a different (not superposed in space) solution (called Solution-2, scores by Phaser RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined in Phenix to R/Rfree 24/26 without any solvent molecule. - The solutions are not superposed in space, although they are near-identical and can be superposed yielding a C-alpha rmd =0.001. - Both structures present VERY similar density maps. The maps are not superposed in space, but when you run the chain in one map in Coot and do the same in the other it they the present exactly the same features. It is impossible to ignore their similarities. - Both structures and maps present the same origin and unit cell. - If I add to Solution-2 the equivalent solvent molecules of Solution-1 (I did this by superposing Solution-1 to Solution-2 then copy/pasting the solvent molecules), the R/Rfree become 22/24. This is a clear indication that the solutions are related. - I can't find any MR solutions using the same template and space groups P222, P2221 and P21212. How two different sets of phases can yield maps with the same features? What is happening, wrong space group? I have a feeling my lack of experience is the problem. Thank you. Regards, Napo
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
On Mon, Nov 21, 2011 at 10:23 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote: Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor But sometimes (or actually we find quite often) the crystal after soaking freezing is sufficiently non-isomorphous (we sometimes see up tp 10% changes in cell parameters) that RBR just doesn't work, and then you have to fall back on MR. The solution in that case to avoid problems of generating symmetry-related and/or origin-shifted molecules is to do a limited MR search, e.g. rotating/translating each molecule in the a.u. independently by up to 5 deg 5 Ang. from the model (which of course has 100% similarity making it a lot easier). Furthermore, because the number of points sampled is much less one can afford to do the more accurate full 6-D search, as opposed to the usual 3-D RF followed by 3-D TF on each RF solution. So now we do this routinely (we don't even bother with a preliminary RBR). I believe the limited 6-D search can be done with Phaser. Cheers -- Ian
Re: [ccp4bb] Strange spots
Dear All, I posted some odd diffraction late last year consisting of Bragg diffraction spots with a diffuse ring or halo. Along with Richard Welberry at ANU we have now published an explanation for this diffuse scattering. For those that are interested the reference is:- Acta Cryst. (2011). B67, 516-524 [ doi:10.1107/S0108768111037542 ] Diffuse scattering resulting from macromolecular frustration T. R. Welberry, A. P. Heerdegen, D. C. Goldstone and I. A. Taylor Kind Regards David -- David Goldstone, PhD National Institute for Medical Research Molecular Structure The Ridgeway Mill Hill London NW7 1AA
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
Or use Kevin's csymmatch which does wonders on scrambled oligomers that (nearly always, at least in my hands) come out of Phaser in cases like those mentioned by Ian and which do need MR. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ian Tickle [ianj...@gmail.com] Sent: Monday, November 21, 2011 1:02 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase On Mon, Nov 21, 2011 at 10:23 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote: Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor But sometimes (or actually we find quite often) the crystal after soaking freezing is sufficiently non-isomorphous (we sometimes see up tp 10% changes in cell parameters) that RBR just doesn't work, and then you have to fall back on MR. The solution in that case to avoid problems of generating symmetry-related and/or origin-shifted molecules is to do a limited MR search, e.g. rotating/translating each molecule in the a.u. independently by up to 5 deg 5 Ang. from the model (which of course has 100% similarity making it a lot easier). Furthermore, because the number of points sampled is much less one can afford to do the more accurate full 6-D search, as opposed to the usual 3-D RF followed by 3-D TF on each RF solution. So now we do this routinely (we don't even bother with a preliminary RBR). I believe the limited 6-D search can be done with Phaser. Cheers -- Ian
Re: [ccp4bb] how to change DNA bases in pdb to match the target DNA sequence
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei, you could use coot Calculate-Model/Fit/Refine-Simple Mutate also for nucleic acids. It may be worth running geometry idealisation in coot or refmac5 before using the model in MR. Tim On 11/19/2011 09:44 PM, Wei Shi wrote: Hi all, I want to change a piece of DNA in pdb file to match the target DNA sequence in order to be used as a search model in molecular replacement. Does anyone happen to know how I could do this? Thank you so much! Best, Wei - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFOyjTzUxlJ7aRr7hoRApYSAJ4xf8R1kjy+sl16WlQRGuOZuW4kbQCbBwB7 hY5x91qUynn4xFxrYHsN/kY= =HaRr -END PGP SIGNATURE-
Re: [ccp4bb] help with the structures
I think you are proving yet again that refinement at 3.3A is not easy. Indeed there are probably multiple conformations for parts of the structure and that may well be why your data is at low resolution and anisotropic. Maybe this is the best you can do.. I think I would make sure the apo structure is as good as it can be, then fit that to the 3.3A data set, and only use that 3.3A data to deduce whatever features differ from the APO structure. Eleanor On 11/19/2011 12:09 PM, Rajesh kumar wrote: Dear All, We have an anisotropic dataset of 3.3 A and it was solved (not by me) with P6522 with R/freeR 29.1/37.3. I got the corrected mtz file by plugging in the .HKL (P6122) file to anisotropy diffraction server at 2.04 A. I reindexed this p6122 to p6522 and extended the resolution and refined (refmac) the structure to R/freeR 36.40/38.50. With aotoncs option, fixing all Ramachnadran and rotamer outliers I got it 30/32. When I added waters and it went down to 27.5/31.2. At this point I recognized that my new .mtz file from anisotropy server has different R flag than the earlier one (3.3A) so I copied the R flag and did refinemnt to get R/Rfree 0.2682/0.3247. When I looked at the refined structure I found more outliers than I fixed in earlier round. I did fix all the outliers and without NCS and waters it gives R/Rfree 0.2906/0.3325. At all the stages I look at outliers at molprobity server which suggested structure is 10th percentile and after refinement more outliers comes back. At stage-1 map looked far better so was happy that anisotropy correction has worked for me (this was my first time handling this type of dataset) but further refinement didn’t make it look any better.I use both refmac and autoBuster for refinement. http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048657095/ This protein is an human enzyme and a bacterial homologue which has 38% identity has been used to solve the Apo structure (2.7 A, pC2221, R/freeR 23.03/27.96, molprobity is around 50th percentile). I looked in to this I try to fix all the outliers and try to improve molprobity score but it just refused to improve as after refinement I get more outliers. This Apo structure was used to solve the mutant structure at 3.3 A. I believe that both structure could have better R/freeR and excellent molprobity scores than what they have now. I am not able to recognise if there is any problem in Apo structure and if errors have come to mutant so both of them refuse to improve. I wondered if there is any model bias (I don’t know if it’s the case but nothing was coming to my mind) so thought using ARP/wARP classic to build model from existing model but it complained that The wilson plot is very bad and ARp/wARP is very unlikely to run in a sensible way. Please check your data . http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048687955/ At this point I dont know how to systematically dissect this problem. I know there could be wrong in several places but with my only '2-3 structure experience' I am not able to identify the regions to look for error but I think something is not right. I really appreciate if you give me some suggestions/ideas/directions/tips so that I could recognize problem and improve structure and learn some more. I appreciate your valuable time. Regards,Rajesh
Re: [ccp4bb] adxv
Dear Mark, $ locate XKeysymDB - didnt come with any thing suggests probably openmotif lib is not installed. I linux server has Fedora and I am using latest version of Adxv so details on the sbgrid suggest its same problem. So how would I fix this. Thanks for your time. Regards Rajesh Date: Sun, 20 Nov 2011 23:18:32 + Subject: Re: [ccp4bb] adxv From: mark.x.bro...@gmail.com To: ccp4...@hotmail.com CC: CCP4BB@jiscmail.ac.uk Dear Rajesh, Are you using the Openmotif library? If so, do you have an XKeysymDB file installed? In as shell, issue: $ locate XKeysymDB You may need to symlink it to /usr/X11R6/lib/X11/XKeysymDB if it is elsewhere [1]. I suspect you're not the only one to have seen this [2]. I hope this helps. Mark [1] ftp://ftp.parallelgeo.com/SPW_Products/Linux/Current_Release/ReadMe_for_recent_Linux_distributions.txt [2] http://sbgrid.org/news/newsletters/2009/06 (search for the string adxv) On 20 November 2011 19:45, Rajesh kumar ccp4...@hotmail.com wrote: Dear Tim, Thanks. Your suggestion of adding to PATH works and its not completely functional may be due to the Ximg/ssh or some thing to do with display. All three windows opened but couldn't open any image as it didn't display in the list of autoload window. Pattern shows *.0. $adxv pin8_1_180.img (standard_in) 1: parse error (standard_in) 1: parse error (standard_in) 1: parse error beam_center x = pixels , mm beam_center y = pixels , mm distance = mm overload = counts pixelsize = 0.172 mm wavelength = Angstroem Adxv Version 1.9.4-beta Copyright (C) 1994-2007 by Andrew Arvai, Area Detector Systems Corporation Using 24-bit TrueColor visual Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: ManagerParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfBeginLine Warning: ... found while parsing ':KeyosfBeginLine: ManagerGadgetTraverseHome()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfHelp Warning: ... found while parsing ':KeyosfHelp: ManagerGadgetHelp()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: ManagerParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfHelp Warning: ... found while parsing ':KeyosfHelp:Help()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfCancel Warning: ... found while parsing ':KeyosfCancel: MenuEscape()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfSelect Warning: ... found while parsing ':KeyosfSelect: ArmAndActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: Locale not supported for XmbTextListToTextProperty Warning: Cannot convert XmString to compound text Warning: translation table syntax error: Unknown keysym name: osfPrimaryPaste Warning: ... found while parsing ':m KeyosfPrimaryPaste:cut-primary()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfSelect Warning: ... found while parsing ':KeyosfSelect: ArmAndActivate()' Warning: String
Re: [ccp4bb] improve protein-DNA complex crystal
my DNA contains a 9-1-9 palindromic sequence,i tried different blunt end DNA,and got two kind of crystal with different lenght DNA.both don't diffract. 2011/11/21 Michael Murphy pn1...@gmail.com Deng, could you tell us a bit more about the DNA that you used? I think you may want to try to optimize the DNA that you are using. Maybe try either a couple base pair longer/shorter piece of DNA? or try switching from blunt ended DNA to a one or two base pair overhang piece of DNA? (very often it is contacts between the ends of the pieces of DNA that drive crystallization) On Mon, Nov 21, 2011 at 2:36 AM, dengzq1987 dengzq1...@gmail.com wrote: ** Hi all, I got a protein-DNA complex crystal,by running SDS-PAGE and nativePAGE,i prove that the crystal contain both protein and DNA.BUT it does not diffract. does anyone have suggestion to improve the diffraction ability ? Best wishes, deng
[ccp4bb] Protein-DNA complex crystallization
Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.* coli* ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S
Re: [ccp4bb] Crystallization plates - 24 well
Greiner Bio One is an alternative with distributors in various different countries worldwide http://www.greinerbioone.com/en/france/articles/catalogue/article-groups/11_5/ On Fri, 18 Nov 2011 12:40:56 -0500, Poorva Dharkar wrote: Hi, I need to buy 24-well crystallization plates. I want to know the review of crystallization plates other than Hampton research Molecular Dimensions (cheaper still good to work with). Has anyone used liked the ones from Jena biosciences? Thank you, Poorva Dharkar National Cancer Institute Building 37 Room 2128 37 Convent Drive Bethesda, MD 20892-4255 -- Irene Margiolaki Lecturer, Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, GR-26500, Patras, Greece Tel: +302610997408 Web site: http://www.biology.upatras.gr/index.php?option=com_contentview=categorylayout=blogid=83Itemid=99 Visiting Scientist, European Synchrotron Radiation Facility (ESRF), Grenoble, France
Re: [ccp4bb] Protein-DNA complex crystallization
Curious, how did you assess that your crystals only have DNA? F On Nov 21, 2011, at 8:59 AM, umar farook wrote: Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.coli ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Protein-DNA complex crystallization
Sent from my Verizon Wireless Phone - Reply message - From: umar farook umarfaroo...@gmail.com Date: Mon, Nov 21, 2011 10:59 am Subject: [ccp4bb] Protein-DNA complex crystallization To: CCP4BB@JISCMAIL.AC.UK Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.* coli* ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S
Re: [ccp4bb] Protein-DNA complex crystallization
What is Kd? Also, in reply to earlier posts: it is sadly common in crystallizing large protein-DNA complexes to go through a couple dozen different duplexes and several dismally-diffracting crystal forms before finding a good one. Phoebe From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of umar farook umarfaroo...@gmail.com) Subject: [ccp4bb] Protein-DNA complex crystallization To: CCP4BB@JISCMAIL.AC.UK Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.coli ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S
Re: [ccp4bb] 1.95A, different phases, maps look the same, R/Rfree 22/24, wrong space group? - RESOLVED
The problem was resolved with your help, and in fact there was no problem, except that I was unaware of the alternative origins. I promise I'm reading more about it. :$ A side note: I collected another crystal, different crystallization condition but same cell parameters, and followed Eleanor Dodson suggestion, just did a rigid body and the map looks just like the others. Thanks you for all the answers, I'm learning a lot by trying to follow (and understand) your different suggestions, and by following this list in general. It is a good reading. Best regards, Napo On 11/20/2011 1:57 PM, Napoleão Valadares wrote: Thank you all for the replies. Felix Frolow, Dan Leahy, Hans Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot. I think I understand it now, I always thought the one ring to rule them all translated in the crystallography realms to one origin to rule them all. That probably means I have a long road in front of me. I'm still half confused, I definitely need to read more, as much as I read about symmetry and space groups I never seem to improve or get a better understanding, but I'll keep trying. About the same origin: The pdbs of both Solution-1 and Solution-2 present the same space group and cell, as observed opening the pdbs as text files or in pymol. When I open both maps on coot they are not superposed but present the same cell and origin. If I open both solutions on pymol they clash. If I generate the symmetry mates of both solutions none of them are superposed, instead they clash. But I think they are related as you all pointed, I'll check it out. Thank you all for your kind answers and your patience with a beginner. Regards from a sunny Brazil, Napo On 11/20/2011 2:58 AM, Felix Frolow wrote: Napoleao, It is so called alternative origins play a game with you. You do not change your structure by shifting 1/2 translation (or even combination of these translations) into directions of the main axes of your unit cell. Structure factors after this operation stay the same, however phases change systematically, producing however the same map features. Would I be a begin crystallographer now, I would read a bit more old fashioned books on crystallography such as probably Jensen and Stout... FF 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 mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote: Hello, I'm observing a very strange phenomena (at least to me, I'm a beginner). It is related to symmetry (I think). I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas 35% in the last shell) and a partially refined solution with R/Rfree 22/24, 166 aminoacids observed and around 30 solvent molecules. I'll call this Solution-1. The refinement was smooth, the densities were very clearly asking for the correct missing side chains and the map looks good. The space group I'm using is P212121, pointless and XDS agree with that (but me and pointless both have a long history of being wrong about space groups). Phenix.xtriage says there's no twinning. I took Solution-1 and used it as a template in a molecular replacement in the same space group (P212121) using the same mtz as the one used to refine the template. I got a different (not superposed in space) solution (called Solution-2, scores by Phaser RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined in Phenix to R/Rfree 24/26 without any solvent molecule. - The solutions are not superposed in space, although they are near-identical and can be superposed yielding a C-alpha rmd =0.001. - Both structures present VERY similar density maps. The maps are not superposed in space, but when you run the chain in one map in Coot and do the same in the other it they the present exactly the same features. It is impossible to ignore their similarities. - Both structures and maps present the same origin and unit cell. - If I add to Solution-2 the equivalent solvent molecules of Solution-1 (I did this by superposing Solution-1 to Solution-2 then copy/pasting the solvent molecules), the R/Rfree become 22/24. This is a clear indication that the solutions are related. - I can't find any MR solutions using the same template and space groups P222, P2221 and P21212. How two different sets of phases can yield maps with the same features? What is happening, wrong space group? I have a feeling my lack of experience is the problem. Thank you. Regards, Napo
[ccp4bb] Movements of domains
Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Movements of domains
I believe ESCET was designed to answer your kind of question Best Roberto On 21 Nov 2011, at 22:03, Filip Van Petegem filip.vanpete...@gmail.commailto:filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: mailto:filip.vanpete...@gmail.com filip.vanpete...@gmail.commailto:filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Movements of domains
Just to clarify: I think the question is about the mathematical sense of significance, and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
- Forwarded Message - From: Michael Thompson mi...@chem.ucla.edu To: e dodson e.dod...@ysbl.york.ac.uk Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase A question regarding the plea for less MR (which I support): There have been several recent instances in which I have used the solution of an isomorphous structure to do rigid body refinement for a new crystal (as described by Eleanor). It has always produced good results. My question is about how to best handle the free set of reflections when doing this? I have heard a number of differing opinions about whether or not it is important to carry the freeR flags from the original structure over to the new data set. I have heard equally convincing arguments from both sides, so my young and impressionable mind does not know who to believe. I was hoping I could get an opinion from the advocates for less MR. Sorry for hijacking this thread, but hopefully it will provide some insight that is relevant to the original post. Thanks! Mike - Original Message - From: Eleanor Dodson c...@ysbl.york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor On 11/20/2011 03:57 PM, Napoleão Valadares wrote: Thank you all for the replies. Felix Frolow, Dan Leahy, Hans Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot. I think I understand it now, I always thought the one ring to rule them all translated in the crystallography realms to one origin to rule them all. That probably means I have a long road in front of me. I'm still half confused, I definitely need to read more, as much as I read about symmetry and space groups I never seem to improve or get a better understanding, but I'll keep trying. About the same origin: The pdbs of both Solution-1 and Solution-2 present the same space group and cell, as observed opening the pdbs as text files or in pymol. When I open both maps on coot they are not superposed but present the same cell and origin. If I open both solutions on pymol they clash. If I generate the symmetry mates of both solutions none of them are superposed, instead they clash. But I think they are related as you all pointed, I'll check it out. Thank you all for your kind answers and your patience with a beginner. Regards from a sunny Brazil, Napo On 11/20/2011 2:58 AM, Felix Frolow wrote: Napoleao, It is so called alternative origins play a game with you. You do not change your structure by shifting 1/2 translation (or even combination of these translations) into directions of the main axes of your unit cell. Structure factors after this operation stay the same, however phases change systematically, producing however the same map features. Would I be a begin crystallographer now, I would read a bit more old fashioned books on crystallography such as probably Jensen and Stout… FF 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 mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote: Hello, I'm observing a very strange phenomena (at least to me, I'm a beginner). It is related to symmetry (I think). I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas 35% in the last shell) and a partially refined solution with R/Rfree 22/24, 166 aminoacids observed and around 30 solvent molecules. I'll call this Solution-1. The refinement was smooth, the densities were very clearly asking for the correct missing side chains and the map looks good. The space group I'm using is P212121, pointless and XDS agree with that (but me and pointless both have a long history of being wrong about space groups). Phenix.xtriage says there's no twinning. I took Solution-1 and used it as a template in a molecular replacement in the same space group (P212121) using the same mtz as the one used to refine the template. I got a different (not superposed in space) solution (called Solution-2, scores by Phaser RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined in Phenix to R/Rfree 24/26 without any solvent molecule. -
Re: [ccp4bb] Movements of domains
Hello Jacob, that's correct, I'm only looking at the mathematical significance, not the biological one. I follow the same reasoning - it is highly improbably for all atoms to be skewed in the same direction. In a case I'm currently looking at, I'm particularly dealing with cryo-EM data, not X-ray structures, but with the same underlying principles: what are the odds that all pixels of the map move together in the same direction? As mentioned for X-ray structures, a Luzzati analysis may give information about the positional errors, but there should be an increased resolution when comparing domain movements, because it's unlikely for all atoms to have an error in the same direction. Filip On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Just to clarify: I think the question is about the mathematical sense of significance, and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Movements of domains
On Nov 21, 2011, at 3:04 PM, Filip Van Petegem wrote: So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? I can think of a different but related question. How significant is a particular movement compared to a measured coordinate error? One way to measure the coordinate error in this example is to least-squares superpose the two instances of the domain in question and calculate the rmsd. This makes the calculation of significance independent of the resolution of the data set. James
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
Hi Mike, Often, I generate independent freeR set (especially in cases where soak dataset is of different resolution (usually worse) compared to the native dataset and do following two things to get rid-off the bias: 1. add a noise to the coordinates (this can be done using PDBSET). 2. set the Bvalues to the wilson B of the soak dataset (within Refmac5 before rigidbody refinement). HTH, Partha On Mon, Nov 21, 2011 at 5:47 PM, Michael Thompson mi...@chem.ucla.eduwrote: - Forwarded Message - From: Michael Thompson mi...@chem.ucla.edu To: e dodson e.dod...@ysbl.york.ac.uk Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase A question regarding the plea for less MR (which I support): There have been several recent instances in which I have used the solution of an isomorphous structure to do rigid body refinement for a new crystal (as described by Eleanor). It has always produced good results. My question is about how to best handle the free set of reflections when doing this? I have heard a number of differing opinions about whether or not it is important to carry the freeR flags from the original structure over to the new data set. I have heard equally convincing arguments from both sides, so my young and impressionable mind does not know who to believe. I was hoping I could get an opinion from the advocates for less MR. Sorry for hijacking this thread, but hopefully it will provide some insight that is relevant to the original post. Thanks! Mike - Original Message - From: Eleanor Dodson c...@ysbl.york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor On 11/20/2011 03:57 PM, Napoleão Valadares wrote: Thank you all for the replies. Felix Frolow, Dan Leahy, Hans Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot. I think I understand it now, I always thought the one ring to rule them all translated in the crystallography realms to one origin to rule them all. That probably means I have a long road in front of me. I'm still half confused, I definitely need to read more, as much as I read about symmetry and space groups I never seem to improve or get a better understanding, but I'll keep trying. About the same origin: The pdbs of both Solution-1 and Solution-2 present the same space group and cell, as observed opening the pdbs as text files or in pymol. When I open both maps on coot they are not superposed but present the same cell and origin. If I open both solutions on pymol they clash. If I generate the symmetry mates of both solutions none of them are superposed, instead they clash. But I think they are related as you all pointed, I'll check it out. Thank you all for your kind answers and your patience with a beginner. Regards from a sunny Brazil, Napo On 11/20/2011 2:58 AM, Felix Frolow wrote: Napoleao, It is so called alternative origins play a game with you. You do not change your structure by shifting 1/2 translation (or even combination of these translations) into directions of the main axes of your unit cell. Structure factors after this operation stay the same, however phases change systematically, producing however the same map features. Would I be a begin crystallographer now, I would read a bit more old fashioned books on crystallography such as probably Jensen and Stout… FF 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 mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote: Hello, I'm observing a very strange phenomena (at least to me, I'm a beginner). It is related to symmetry (I think). I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas 35% in the last shell) and a partially refined solution with R/Rfree 22/24, 166 aminoacids observed and around 30 solvent molecules. I'll call this Solution-1. The refinement was smooth, the densities were very clearly asking for the correct missing side chains and the map looks good. The space group I'm using
Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
I'll jump in here, and avoid the question entirely. Since running MR on an isomorphous crystal gives the same answer as just stuffing in the model and running some rigid body refinement, however you decide to handle your R free flags, you should do the same thing in both cases. The model is the same. Dale Tronrud On 11/21/11 14:47, Michael Thompson wrote: - Forwarded Message - From: Michael Thompson mi...@chem.ucla.edu To: e dodson e.dod...@ysbl.york.ac.uk Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase A question regarding the plea for less MR (which I support): There have been several recent instances in which I have used the solution of an isomorphous structure to do rigid body refinement for a new crystal (as described by Eleanor). It has always produced good results. My question is about how to best handle the free set of reflections when doing this? I have heard a number of differing opinions about whether or not it is important to carry the freeR flags from the original structure over to the new data set. I have heard equally convincing arguments from both sides, so my young and impressionable mind does not know who to believe. I was hoping I could get an opinion from the advocates for less MR. Sorry for hijacking this thread, but hopefully it will provide some insight that is relevant to the original post. Thanks! Mike - Original Message - From: Eleanor Dodson c...@ysbl.york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase Just a plea for less molecular replacement. If you get a new crystal of a known protein with the same cell dimension as youur old crystal, the most likely scenario is that it has the same group, and you really should not try MR - use the previous solution as input to do rigid body refinement, and then a) the R factor will tell you if this is a reasonable hypothesis (it usually is..) and b) you dont have this awful problem of not being able to compare the solutions.. Eleanor On 11/20/2011 03:57 PM, Napoleão Valadares wrote: Thank you all for the replies. Felix Frolow, Dan Leahy, Hans Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot. I think I understand it now, I always thought the one ring to rule them all translated in the crystallography realms to one origin to rule them all. That probably means I have a long road in front of me. I'm still half confused, I definitely need to read more, as much as I read about symmetry and space groups I never seem to improve or get a better understanding, but I'll keep trying. About the same origin: The pdbs of both Solution-1 and Solution-2 present the same space group and cell, as observed opening the pdbs as text files or in pymol. When I open both maps on coot they are not superposed but present the same cell and origin. If I open both solutions on pymol they clash. If I generate the symmetry mates of both solutions none of them are superposed, instead they clash. But I think they are related as you all pointed, I'll check it out. Thank you all for your kind answers and your patience with a beginner. Regards from a sunny Brazil, Napo On 11/20/2011 2:58 AM, Felix Frolow wrote: Napoleao, It is so called alternative origins play a game with you. You do not change your structure by shifting 1/2 translation (or even combination of these translations) into directions of the main axes of your unit cell. Structure factors after this operation stay the same, however phases change systematically, producing however the same map features. Would I be a begin crystallographer now, I would read a bit more old fashioned books on crystallography such as probably Jensen and Stout… FF 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 mailto:mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote: Hello, I'm observing a very strange phenomena (at least to me, I'm a beginner). It is related to symmetry (I think). I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas 35% in the last shell) and a partially refined solution with R/Rfree 22/24, 166 aminoacids observed and around 30 solvent molecules. I'll call this Solution-1. The refinement was smooth, the densities were very clearly asking for the correct missing side chains and the map looks good. The space group I'm using is P212121, pointless and XDS agree with that (but me and pointless both have a long history of being wrong about space groups). Phenix.xtriage
Re: [ccp4bb] Movements of domains
This is a subtle problem and performing an analysis of this type of error is confusing. Most of the tools we use to analyze errors begin with the assumption that the errors are random and uncorrelated. These include Luzzati and Fo-Fc maps. My solution is to perform a null hypothesis test. If you run two refinements starting from the same model, in one allowing the RB shift and in the other forbidding it, which fits the data better? If the difference in likelihood is quite small then you cannot distinguish between a RB shifted model and one w/o the shift and that shift must be insignificant (in a statistical sense.) If the likelihood is better when the shift is allowed then the shift is significant. In my experience RB shifts of a couple tens of an Angstrom are very significant even with 4 A resolution data. X-ray diffraction is exquisitely sensitive to this sort of motion. Dale Tronrud On 11/21/11 14:52, Filip Van Petegem wrote: Hello Jacob, that's correct, I'm only looking at the mathematical significance, not the biological one. I follow the same reasoning - it is highly improbably for all atoms to be skewed in the same direction. In a case I'm currently looking at, I'm particularly dealing with cryo-EM data, not X-ray structures, but with the same underlying principles: what are the odds that all pixels of the map move together in the same direction? As mentioned for X-ray structures, a Luzzati analysis may give information about the positional errors, but there should be an increased resolution when comparing domain movements, because it's unlikely for all atoms to have an error in the same direction. Filip On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller j-kell...@fsm.northwestern.edu mailto:j-kell...@fsm.northwestern.edu wrote: Just to clarify: I think the question is about the mathematical sense of significance, and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 tel:%2B1%20604%20827%204267 email: filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email:
[ccp4bb] sugar and coot
Hi everyone! Does anyone know if there is a way of auto-refining a sugar in Coot? Jan
Re: [ccp4bb] Movements of domains
On Nov 21, 2011, at 3:52 PM, Filip Van Petegem wrote: As mentioned for X-ray structures, a Luzzati analysis may give information about the positional errors, but there should be an increased resolution when comparing domain movements, because it's unlikely for all atoms to have an error in the same direction. Here's how I think about it: If you use the empirical coordinate error that I described previously, you can use simple statistics to calculate how likely you are to get a coordinated movement (relative to a fixed landmark). I can use a 1-d case as an example. In this 1-d case, let's pretend that we have a domain of N=25 atoms where atom 2 is about 1 away from atom 1 and atom 3 is 2 away from atom 1 and one away from atom 2, etc, with a standard deviation of 1 for the position of the atoms. If atom 1 for domain A is at 1, this is just A_j = j Then you can have domain B that has moved +1 compared to domain A: B_j = j+1 Since we have an alignment (B_j - A_j), then we can calculate the movement, X: X = mean(B) - mean(A) We can also calculate the error of the ensemble (aka the error of the mean): sigmaE = std( (B - mean(B)) - (A - mean(B)) ) / sqrt(25) Then, we can calculate how likely it is we observe the movement X by tail integration of the cumulative normal distribution. We will justify this for the 3-d case because the least squares superposition (from which we estimate the coordinate error) assumes normality. Here is a simulation of this scenario in python: py import numpy py from scipy.special import ndtr py a = numpy.array([numpy.random.normal(j) for j in xrange(25)]) py b = numpy.array([numpy.random.normal(j+1) for j in xrange(25)]) py a array([ 1.38125295, -0.27126096, 1.7597104 , 1.36242299, 3.88327659, 4.33063307, 5.00544708, 7.0258, 7.83945228, 9.72101719, 10.36231633, 10.29176378, 11.78497375, 12.16082056, 14.31057296, 13.25941344, 17.93779336, 18.05626047, 18.62148347, 20.52756478, 19.73362283, 21.83953268, 22.28038617, 23.24545481, 22.96192518]) py b array([ 3.32750181, 2.42664791, 3.23309368, 4.32882699, 6.59985764, 6.49597664, 5.27921723, 7.8573831 , 9.98722475, 10.65225383, 11.69970159, 11.67435798, 12.16191254, 13.69297801, 14.21845382, 17.21423427, 16.89347161, 17.68778305, 17.89371115, 18.7679351 , 20.84842496, 20.69249899, 23.97436807, 23.54011453, 26.84986504]) py X = b.mean() - a.mean() py sigma_ensemble = ((b - b.mean()) - (a - a.mean())).std() / math.sqrt(25) py X_standardized = (X - 0) / sigma_ensemble py 2 * ndtr(-abs(X_standardized)) 0.00011596192653578624 This means, for the 1-d scenario I describe, (using the random arrays generated above), the movement is expected about once for every 10,000 experiments, providing a p-value, or estimate of significance. Note that the 2 comes from the fact that the cumulative distribution has 2 tails. A 3-D calculation using the rmsd as the coordinate error would be similar except that you use Euclid's formula to calculate the distances in higher dimensions (instead of the absolute value of a simple subtraction as in 1-d). James
[ccp4bb] Coot, RH6.1 x86 linux
I just installed CCP4-6.2.0 and COOT on a Red Hat v6.1, x86, linux workstation. Some of the menus, along the right edge, are gray buttons on gray background. Where is the preferences file to change the colors of the menu buttons? The menus across the top of the window are fine. Also, idiffdisp is unable to view a file. It loads the idiffdisp window, but gives error messages. Is there a log file that I can scan? I think I'm missing some library files for x86. Bernie -- Bernard D. Santarsiero Research Professor Center for Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy Center for Structural Biology Center for Clinical and Translational Science University of Illinois at Chicago MC870 3070MBRB 900 South Ashland Avenue Chicago, IL 60607-7173 USA (312) 413-0339 (office) (312) 413-9303 (FAX) http://www.uic.edu/labs/bds
Re: [ccp4bb] Movements of domains
On Nov 21, 2011, at 5:23 PM, James Stroud wrote: except that you use Euclid's formula to calculate the distances in higher dimensions I meant to say Euclidian distance. Euclid's formula has a specific meaning that is different.
Re: [ccp4bb] sugar and coot
Hi, I'm not sure if that is what you are looking for, but if you want to use the real space refine zone function in coot with sugars that is possible. For a monosaccharide you will need a cif dictionary with the restraint definitions - in some cases present in the refmac monomer database, but be careful not all cif files there are correct. If none is present or if your monomer is disrupted during refinement you can look at the small molecule databases (Hic-Up etc) or calculate one with PRODRG (http://davapc1.bioch.dundee.ac.uk/prodrg/). Load it with File Import CIF dictionary and real space refinement should work. If you try to refine a polysaccharide it's a bit more difficult since coot will ignore any glycosidic bond restraints - if two sugars are close by and one has well defined density and the other one not it might even place them on top of each other. I like to use the anchor tool on the bonding atoms in those cases. The other option for a polysaccharide is to use PRODRG to generate a single monomer out of your polysaccharide. In any case at the end you have to inspect the geometry manually since the restraint definitions are often incomplete. Greetings Dirk Am 22.11.11 01:16, schrieb Jan van Agthoven: Hi everyone! Does anyone know if there is a way of auto-refining a sugar in Coot? Jan
Re: [ccp4bb] Movements of domains
I am curious how all of this can be more than splitting hairs, i.e., under what conditions can this 1Ang domain motion mean something biologically significant? Proteins are pretty flexible, after all, especially between domains. JPK On Mon, Nov 21, 2011 at 6:41 PM, James Stroud xtald...@gmail.com wrote: On Nov 21, 2011, at 5:23 PM, James Stroud wrote: except that you use Euclid's formula to calculate the distances in higher dimensions I meant to say Euclidian distance. Euclid's formula has a specific meaning that is different. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Movements of domains
On Nov 21, 2011, at 6:34 PM, Jacob Keller wrote: I am curious how all of this can be more than splitting hairs, i.e., under what conditions can this 1Ang domain motion mean something biologically significant? To engage in the discussion, I think we had to accept this: On Nov 21, 2011, at 3:04 PM, Filip Van Petegem wrote: I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the point of the discussion, as I understand it, is to figure out whether the movement warrants further consideration in the first place, i.e. whether it is significant with respect to the error of the models. I think it doesn't take too much energy to discount the attempt to quantify the statistical significance by claiming that one can't imagine how such a change might be biologically significant. I'm really not privy to the structures in question, so I am in no position to make this judgement. James
Re: [ccp4bb] Movements of domains
If the difference in likelihood is quite small then you cannot distinguish between a RB shifted model and one w/o the shift and that shift must be insignificant (in a statistical sense.) If the likelihood is better when the shift is allowed then the shift is significant. That of course is correct and leads to the interesting (and in part previously discussed) question how to quantify (log) likelihood ratios in terms of significance. I am not sure that this is trivial. More likely better, very likely better, kinda really likely better? Having said that I also want to caution the normal distribution statistical test fans, who think that a p value or similar has any more meaning. Whether p=0.05 means something (other than a statistical metric) is equally fuzzy; it just conveys a false sense of precision and erudition. May I quote: The scientist must be the judge of his own hypotheses, not the statistician. A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept of likelihood and its application to scientific inference , p. 34. Brrr On 11/21/11 14:52, Filip Van Petegem wrote: Hello Jacob, that's correct, I'm only looking at the mathematical significance, not the biological one. I follow the same reasoning - it is highly improbably for all atoms to be skewed in the same direction. In a case I'm currently looking at, I'm particularly dealing with cryo-EM data, not X-ray structures, but with the same underlying principles: what are the odds that all pixels of the map move together in the same direction? As mentioned for X-ray structures, a Luzzati analysis may give information about the positional errors, but there should be an increased resolution when comparing domain movements, because it's unlikely for all atoms to have an error in the same direction. Filip On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller j-kell...@fsm.northwestern.edu mailto:j-kell...@fsm.northwestern.edu wrote: Just to clarify: I think the question is about the mathematical sense of significance, and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 tel:%2B1%20604%20827%204267 email: filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Movements of domains
A mixture between mathematical significance and biological significance as a part of the reply: you should also take into account the thermal vibrations of the atoms present in that domain, i.e. the thermal ellipsoids when you have one of the representations of anisotropic temperature factors (when these can be obtained, high enough resolution), together with the associated density smearing. Especially if you observe correlated thermal ellipsoids. If you have a small motion but that this motion can be (at least in good part) explained by the inherent thermal flexibility of all atoms in that domain then perhaps you can question the significance of this domain motion (at least in the publication). Fred. Filip Van Petegem wrote: Dear crystallographers, I have a general question concerning the comparison of different structures. Suppose you have a crystal structure containing a few domains. You also have another structure of the same, but in a different condition (with a bound ligand, a mutation, or simply a different crystallization condition,...). After careful superpositions, you notice that one of the domains has shifted over a particular distance compared to the other domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now how can you know that this is a 'significant' change? Say the overall resolution of the structures is lower than the observed distance (2.5A for example). Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at this resolution would seem wrong: we're not talking about some electron density protruding a bit more in one structure versus another, but all of the density has moved in a concerted fashion. So this would seem 'real', and not due to noise. I'm not talking about the fact that this movement was artificially caused by crystal packing or something similar. Just for whatever the reason (whether packing, pH, ligand binding, ...), you simply observe the movement. So the question is: how you can state that a particular movement was 'significantly large' compared to the resolution limit? In particular, what is the theoretical framework that allows you to state that some movement is signifcant? This type of question of course also applies to other methods such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a 10A map? If it is, how do we quantify the significance? If anybody has a great reference or just an individual opinion, I'd like to hear about it. Regards, Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com mailto:filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
