Re: [ccp4bb] validating a homlology model
Assuming that your homology model is that of a dimer, you could put it in a large unit cell (just add CRYST1 record). The only interface you will get from pisa will be your dimer interface. If your homology model is a monomer, then pisa will not help, of course, and you would need to predict dimerization mode first. Happy Connecting. Sent from my Sprint Samsung Galaxy S® 5 Original message From: Careina Edgooms <02531c126adf-dmarc-requ...@jiscmail.ac.uk> Date: 3/2/18 6:44 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] validating a homlology model Dear all What programs are best used for validate homology models? I know of molprobity but if there are no coordinates I cannot use it. Is there a way to use such programs with homology models? Also I wish to use pdbepisa for to charaterise dimer interface but again for homology model this cannot be done as there is no PDB model. Does anybody know way to use PISA software on my own model that is not deposited in PDB? Thank you in advanceCareina
Re: [ccp4bb] question related to MTZ file
I suspect the question might be about converting intensities to anplitudes. If so ctruncate is your friend. Happy Connecting. Sent from my Sprint Samsung Galaxy S® 5 Original message From: Eleanor Dodson eleanor.dod...@york.ac.uk Date:01/03/2015 3:42 PM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] question related to MTZ file I don't really understand your question. An mtz file is simply a format to hold at least a list of h k l Observations Sigma_observations It is a binary file format which makes it very suitable to store information with a large range of value - viz, observations.. Then other information pertinent to reflection indices can be added to the file e.g. IF you have a model you could calculate Fcalc and PHIcalc. and append then IF you had some experimental information to help you determine phases such as anomalous measurements you may want to store these, plus the derived phases and Figs_of_Merit. Is this any help? Phases have to be based on extra information.. Eleanor On 3 January 2015 at 04:00, luzuok luzuo...@126.com wrote: How initial this MTZ file? Does this mtz file contain phase information? Lu Zuokun -- 卢作焜 南开大学新生物站A202 在 2015-01-02 00:52:53，Jurgen Bosch jbos...@jhu.edu 写道： I assume you mean with initial mtz file the one derived from Mosflm after processing your images. If so, then how about running that file through scala http://www.ccp4.ac.uk/html/scala.html ? I bet there are some tutorials on the CCP4 webpage on that topic. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Dec 31, 2014, at 9:19 PM, Dialing Pretty 03f1d08ed29c-dmarc-requ...@jiscmail.ac.uk wrote: Dear All, After I got the initial MTZ file without the structure factors, will you please tell me by which CCP4 program I can get the MTZ file with the structure factors? I am looking forward to getting your reply. Dialing
Re: [ccp4bb] water at the same exactly position
Why? Merging waters like that in coot is a user error, and it does not happen too often. I realize you are talking about changeable settings, but it would be really annoying if a refinement program kept removing water molecules that were placed manually because it does not fit some internal standard. Adding waters by default may obscure density that is due to other components. I just feel one should be careful when changing chemical compositon of a model. That is something algorithms do not (yet) do well. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Pavel Afonine pafon...@gmail.com /divdivDate:10/30/2014 12:27 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] water at the same exactly position /divdiv /divThis conversation makes me more and more certain that water update (add/remove/refine water) done as part of (a default) refinement run is a good idea -:) Pavel On Wed, Oct 29, 2014 at 5:08 AM, luzuok luzuo...@126.com wrote: Dear all, I found that there are some water molecules in my pdb that share the same position. This maybe cause by merging molecules in coot. It seems that I have mereged water molecules into my protein for more than one time. Does anyone tell me how to fix this problem? Best regards! Lu Zuokun -- 卢作焜 南开大学新生物站A202
Re: [ccp4bb] Merge PDB chains
Edit the ATOM records? Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: luzuok luzuo...@126.com /divdivDate:10/23/2014 6:51 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] Merge PDB chains /divdiv /divDear all, Sorry to ask a simple question. There are many SO4 in my PDB file, one belongs to a different chain. I want to merge them into one chain, can anyone tell me how to do this? Best regards! Lu Zuokun -- 卢作焜 南开大学新生物站A202
Re: [ccp4bb] I222 - P22121 space-group ambiguity
Yes, having different crystal forms in the same crystallization conditions is, while clearly uncommon, not unheard of. I had a case once where at least four different crystal forms were observed in crystals harvested from identically prepared drops. It may be that there is some major set of contacts that promote certain arrangement of protein molecules (fiber-like), and these can then be packed in multiple ways as controlled by another ser of weaker interactions. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Florian Schmitzberger schmitzber...@crystal.harvard.edu /divdivDate:10/13/2014 4:47 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] I222 - P22121 space-group ambiguity /divdiv /divHi everybody, I collected a number of X-ray data sets from crystals originating from the same cryst. drop. I solved the initial structure in P22121 space group by MR with Phaser locating two molecules (data to ~ 2.1 Angstr.); refined R/Rfree: 0.213/0.244. Processing of some of the other data sets with XDS/Aimless is consistent with I222 space group (resolution ~ 2.6 Ang.). I can locate one molecule. The unit-cell dimensions for I222 and the initial P22121 space groups for two of the data sets are: I222: a=87.8 b=101.18 c=123.63; P22121: a=93.34 b=105.47 c=122.98; I superposed the molecule in I222 onto one of the two located for the initially solved P22121; the orientation of the NCS-related molecule in P22121 differs from the crystallographic-symmetry related one in I222. Trying to solve this P22121 data set in I222 with MR, does not result in high Z scores, and maps do not look good. Some of the data sets that process in I222 to ~ 3 Angstr., I can also solve in P22121, locating two molecules (differences may not be that clear in this case, since the resolution is lower). Some other data sets process in P22121 with Aimless; with a substantial off-origin Patterson peak, indicating translational NCS. For these, Phaser positions two molecules that are related by crystallographic translational NCS. These two molecules are crystallographic-symmetry related in the original P22121 data set. I can also solve these data sets in I222 space group, with the overall Z score higher than for the P22121 data. I am uncertain, what the ‘true’ space group for some of my data sets is. Could it be that for data that process in P22121, but can be solved in I222, reflections that would indicate I222 space group were not collected? Alternatively, perhaps what I am seeing is that there is a (gradual) transition of the crystal lattice (between P22121 and I222 or vice versa), caused by variation in crystal handling/cooling or exposure to X-rays. It’s relevant to me, because in P22121 space group, a region of the molecule that is of biological interest makes NCS-related crystal contacts that are crystallographic-symmetry related in I222. Has anybody observed similar cases? I would appreciate comments. Cheers, Florian
[ccp4bb]
Try refining your model both ways (with and without covalent link) and see if electron density maps give you an indication. At this resolution there will be some model bias, so be critical. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: rohit kumar rohit...@gmail.com /divdivDate:08/19/2014 2:56 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] /divdiv /divDear All, i have solved a structure of 3.2 A. That is a PLP depended enzyme. In their resting state, PLP-dependent enzymes are usually joined by a covalent aldimine linkage to an essential lysine residue with a C=N bond. This generates the so-called internal aldimine moiety. Could anybody tell me how we can say that this PLP is covalently attached to the Lys or its making Schiff base or not. -- WITH REGARDS Rohit Kumar Singh Lab. no. 430, P.I. Dr. S. Gourinath, School of Life Sciences, Jawaharlal Nehru University New Delhi -110067
Re: [ccp4bb] cc1/2 value in hkl2000
Probably the only way is to take unmerged scalepack output to aimless. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Faisal Tarique faisaltari...@gmail.com /divdivDate:08/15/2014 9:40 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] cc1/2 value in hkl2000 /divdiv /divHello everyone Can anybody please tell me where to locate the Corelation value between half sets (CC1/2) of a data processed through HKL2000 ?? -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] CC-half value ??
Same here. Ultimately, the KD test must be used in the end to finalize the resolution (keeping in mind recently discussed issues of effective resolution given data completeness). I just want to add that at least some versions of aimless report overestimated resolution based on CC1/2 cutoff when outliers are present (e.g. due to ice rings or salt diffraction). It seems that aimless just picks the highest resolution bin where cc1/2 0.5 even if some lower resolution bins are below 0.5 as well. I have written a script for more robust automated evaluation of these curves. In a nutshell, it fits CC1/2 (d) curve to 1/(1+exp (-x)) and returns the resolution at midpoint. I'm pretty sure that theoretical CC1/2 (d) dependence is different from this, but it seems good enough for a rough estimate. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Roger Rowlett rrowl...@colgate.edu /divdivDate:08/14/2014 5:44 PM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] CC-half value ?? /divdiv /divExactly. Aimless will give you suggested resolution cutoffs based on CC 1/2 in the log file. Roger Rowlett On Aug 14, 2014 5:04 PM, conan仙人指路 conan_...@hotmail.com wrote: Hi Faisal, CC-half standard is valuable in evaluating the cut-off of highest resolution. Sometimes even if I/sigI is close to 1 and completeness is not as high, if CC-half is still significant, it may be worth incorporate the extra high-res shell data and extend the resolution. Again, if only the reliability and unbias are carefully confirmed, and the apparent significant CC-half is not due to an artifact of some other factors like ice ring etc. (Ref: Karplus PA and Diederichs K. 2012 Science 336, 1030-1033 https://www.pubmed.com/pubmed/22628654) It has yet to be appreciated by most population of the crystallography society, unlike the I/sigI, completeness, Rsym. In particular, Rsym has gradually less a direct measurement of the data quality and or determinant of resolution cut-off. Best, Conan Hongnan Cao, Ph.D. Department of Biochemistry Rice University Date: Fri, 15 Aug 2014 01:39:48 +0530 From: faisaltari...@gmail.com Subject: [ccp4bb] CC-half value ?? To: CCP4BB@JISCMAIL.AC.UK Dear all How CC-half value of a data set determines the maximum resolution limit during data processing ?? Although much we know about the Rsym and I/Isig values of the highest resolution shell while processing the data, what are the parameters we need to check related to CC-half values ?? -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] thin shell Rfree set selection
By all means, try it both ways and see whether the R-Rfree gap narrows with random vs thin shell selection. Depending on resolution and data quality, you may also consider imposing NCS restraints. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Xianchi Dong dongxian...@gmail.com /divdivDate:08/12/2014 4:45 PM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] thin shell Rfree set selection /divdiv /divDear all, I have a dataset of C2 symmetry and 2 molecules in the ASU. I am wondering if I have to use thin shell Rfree set selection to avoid Rfree bias by NCS. And which Rfree selection method is better? Thanks in advance. Xianchi
Re: [ccp4bb] The modified DNA could not be linked to the downstream DNA in the COOT?
You may need to 1) modify _chem_comp.group to be DNA in the cif-file 2) import the resulting cif-file in coot Cheers Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Wang, Wei wew...@ucsd.edu /divdivDate:07/15/2014 5:14 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] The modified DNA could not be linked to the downstream DNA in the COOT? /divdiv /divHi, There is a problem about modified DNA refinement. I generated one CIF file of a modified DNA base using eLBOW software of PHENIX. However I found the modified DNA could not be linked to the downstream DNA when I refined it in the COOT. Then I generated another CIF file using sketcher of CCP4, but the problem still existed. Is there any expert to help me? Thanks!! Best Wei
Re: [ccp4bb] emergency substitute for RT loop cover?
Try to put your crystal into oil drop? Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Frank von Delft frank.vonde...@sgc.ox.ac.uk /divdivDate:07/07/2014 12:32 PM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] emergency substitute for RT loop cover? /divdiv /divHi all Pretend you were stuck having to do RT data collection but without access to either Mitegen MicroRT Capillaries or the more old-fashioned quartz capillaries, to pop over the loop. Anybody have suggestions of alternative ways of doing this? I do want to use loops (I never learnt how to suck up crystals in capillaries). I have access to a passably stocked biochemistry teaching lab, and could at a pinch go rifle some more advanced research labs. (No, I'm not at home ;) Thanks! phx
Re: [ccp4bb] Kabat, insertion codes refinement
There is no actual requirement to use Kabat numbering, you can avoid it alrogether. Some argue that L27A is actually 28th amino acid in the protein sequence, and labeling it as L27A is simply incorrect. I would suggest doing refinement with plain numbering (no insertion codes) and changing it only for the final model if needed for comparative analysis. Ed Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Hargreaves, David david.hargrea...@astrazeneca.com /divdivDate:06/16/2014 6:07 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] Kabat, insertion codes refinement /divdiv /divDear CCP4bb, I’m refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these residues in refinement? Thanks, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] metal ion dominating density!
Or, if for whatever reason sphere refine isn't an option, fix the metal and all of its ligands Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Paul Emsley pems...@mrc-lmb.cam.ac.uk /divdivDate:06/05/2014 3:51 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] metal ion dominating density! /divdiv /divOn 05/06/14 08:27, Dean Derbyshire wrote: Hi all, I recall there was a post a while back, but silly me can’t remember the details – How does one stop metal ions dominating real space refinement in COOT? sphere refine? Paul.
Re: [ccp4bb] baverage: no tables were found in this file
This may not work if a program implements its own algorithm for parsing command line parameters Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Tim Gruene t...@shelx.uni-ac.gwdg.de /divdivDate:06/03/2014 8:27 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] baverage: no tables were found in this file /divdiv /div-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Eleanor, yes, it does. At least on Linux and OpenBSD (i.e. 'real' UNIX) typing # mkdir dir with spaces # cd dir\ with\ spaces/ # touch file with spaces # echo Some text file\ with\ spaces causes no problems at all. Changing code reliably to include accents, though, might be much more work than enclosing words with double quotes. Best, Tim On 06/03/2014 02:23 PM, Eleanor Dodson wrote: I thought the addition of ... around file names got round this problem eg This_is_Mine.pdb and That is Yours.pdb Eleanor On 3 June 2014 13:15, Eugene Krissinel eugene.krissi...@stfc.ac.uk wrote: I am afraid that it is us who will need to conform eventually :), same to say about Program Files (x86) and the overall culture in Windows, the system where some 40% () of CCP4 users have chosen to work. Eugene On 3 Jun 2014, at 13:03, Mark J van Raaij wrote: completely agree with avoiding spaces in paths and file names, but Google Drive is very handy for sharing files, so every once in a while I forget to move a file someone shares with me before running Unix programs on it and get strange errors. Depending on how awake one is at the time finding out the source can be time-consuming...Google should really remove the space! Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 13:47, Eugene Krissinel wrote: I take this chance to to confirm once more, as publicly as possible, that file paths with spaces are discouraged in today's CCP4. This inconvenience originates from ancient times in computing when good half of CCP4 was written and when spaces were disallowed on file system nodes. Please take a notice of this fact as CCP4 core still receives (albeit infrequent) bug reports, where surprising behaviour is due to using file paths with white spaces. Fixing this has proved to be a hard problem, purely because of technical choices made quite a number of years ago. But good news are that this limitation will be removed in new CCP4 Gui under development. Eugene On 3 Jun 2014, at 08:23, Mark J van Raaij wrote: This also occurred to me once where the file path had a space,(/Google Drive/), when I moved the file somewhere else it worked. I was using baverage from the CCP4i GUI. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 09:20, Tim Gruene wrote: Dear Bing, can you post the exact command you were using, please? Also please check with a different PDB file. In case you are using baverage from the command line, can you make sure you are actually using the program from ccp4 by typing 'which baverage' at the command prompt? Regards. Tim On 06/02/2014 10:16 PM, Wang, Bing wrote: Hi CCP4, Recently when I input my pdb file and run the baverage in the ccp4 suit to check the temperature factor, it always tell me No tables were fund in this file. Could you tell me how to fix this problem? Or is there another software instead of baverage I could use to check the temperature factor? Thanks! Bing -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Scanned by iCritical. -- Scanned by iCritical. - -- - -- 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/ iD8DBQFTjb8eUxlJ7aRr7hoRApyPAKCGq/1XweO13d57W9vaho+DoXLHSgCeJXYW 7mc+g+F1Win65zZc8+nLN6I= =U7dS -END PGP SIGNATURE-
Re: [ccp4bb] baverage: no tables were found in this file
Would a no-space symlink resolve this? Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Mark J van Raaij mjvanra...@cnb.csic.es /divdivDate:06/03/2014 8:03 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: Re: [ccp4bb] baverage: no tables were found in this file /divdiv /divcompletely agree with avoiding spaces in paths and file names, but Google Drive is very handy for sharing files, so every once in a while I forget to move a file someone shares with me before running Unix programs on it and get strange errors. Depending on how awake one is at the time finding out the source can be time-consuming...Google should really remove the space! Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 13:47, Eugene Krissinel wrote: I take this chance to to confirm once more, as publicly as possible, that file paths with spaces are discouraged in today's CCP4. This inconvenience originates from ancient times in computing when good half of CCP4 was written and when spaces were disallowed on file system nodes. Please take a notice of this fact as CCP4 core still receives (albeit infrequent) bug reports, where surprising behaviour is due to using file paths with white spaces. Fixing this has proved to be a hard problem, purely because of technical choices made quite a number of years ago. But good news are that this limitation will be removed in new CCP4 Gui under development. Eugene On 3 Jun 2014, at 08:23, Mark J van Raaij wrote: This also occurred to me once where the file path had a space,(/Google Drive/), when I moved the file somewhere else it worked. I was using baverage from the CCP4i GUI. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 3 Jun 2014, at 09:20, Tim Gruene wrote: Dear Bing, can you post the exact command you were using, please? Also please check with a different PDB file. In case you are using baverage from the command line, can you make sure you are actually using the program from ccp4 by typing 'which baverage' at the command prompt? Regards. Tim On 06/02/2014 10:16 PM, Wang, Bing wrote: Hi CCP4, Recently when I input my pdb file and run the baverage in the ccp4 suit to check the temperature factor, it always tell me No tables were fund in this file. Could you tell me how to fix this problem? Or is there another software instead of baverage I could use to check the temperature factor? Thanks! Bing -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Scanned by iCritical.
Re: [ccp4bb] negative density around disulfide bond
Try refining without disulfide bond - from the way density looks it might work. Whether this is what happens in vivo is a different question entirely. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Eze Chivi ezech...@outlook.com.ar /divdivDate:06/02/2014 1:08 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] negative density around disulfide bond /divdiv /divHello, when I refine my structure, I see negative density around the disulfide bond. I have 7 copies per ASU, and I can see this density in many of them. In some cases, I see positive density also (negative in the center of the straight line linking S atoms, and positive in both sides). What can I try to solve it? Is it due to radiation damage? Alternative conformation (partial oxidation)? Incorrect disulfide geometry parameters? My resolution is 2.1 A, R/Rfree are around 0.220/0.243, and similar results with refmac5, phenix and PDBREDO. Please find two example pictures in attachment. Thanks for your help! Ezequiel
Re: [ccp4bb] (high) values of R-factors in outermost resolution shell
My suggestion is to ignore Rmerge when making decisions about resolution cutoff. While cc1/2 may still perhaps qualify for the recent discussion tag (is two years recent?), deeply flawed nature of the Rmerge concept is over a decade old. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: sreetama das somon_...@yahoo.co.in /divdivDate:06/02/2014 4:27 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] (high) values of R-factors in outermost resolution shell /divdiv /divDear All, What are reasonable values of Rmerge in the outermost resolution shell? Some of the recent discussions suggest going to those sheels where I/sig(I) ~2 and CC1/2 = 0.5. But I am getting Rmerge Rmeas 1 in the outermost shell for those values of I/sig(I) and CC1/2, and I don't think that makes any sense. Reducing the resolution cut-off while data reduction scaling (aimless) reduces the R-values, but I am not sure how much I should reduce the resolution (if at all). Following are the results from the aimless log file: At a resolution cut-off of 1.62A: Overall InnerShell OuterShell Low resolution limit 42.12 42.12 1.65 High resolution limit 1.62 8.87 1.62 Rmerge (within I+/I-) 0.071 0.019 1.325 Rmerge (all I+ and I-)0.074 0.020 1.381 Rmeas (within I+/I-) 0.078 0.021 1.446 Rmeas (all I+ I-)0.077 0.022 1.441 Rpim (within I+/I-)0.031 0.009 0.575 Rpim (all I+ I-) 0.022 0.007 0.410 Rmerge in top intensity bin0.031- - Total number of observations 311022 1839 14876 Total number unique25311 184 1212 Mean((I)/sd(I)) 19.2 52.7 2.0 Mn(I) half-set correlation CC(1/2) 1.000 1.000 0.740 Completeness 100.0 99.4 100.0 Multiplicity12.3 10.0 12.3 At 1.6A, the I/sig(I) and CC1/2 in the outermost shell are lower, and the R-merge,meas,pim are higher. looking forward to your suggestions, thanking you, sreetama
Re: [ccp4bb] distinguish ligand binding sites within a protein
Baerbel, Certainly, constraints of the crystal lattice affect everything. The magnitude of the influence is what is in question here. Let's do the numbers (this is admittedly simplistic but reflect general trends well). Let's say you observe difference in occupancies for two identical sites that is 50%/75%. This means that difference in binding affinities is due to change in free energy of binding at DDG=RTlog(K1/K2). If ligand is in excess with respect to the protein, K~(1-occ)*L/occ. Thus the estimate of DDG is ~0.6kcal/mol. Point is that it does not take much free energy to result in difference in occupancy. Yet anyone who ever done ITC would tell you that such small difference would be hard to detect with the technique. So the prior is that Wei sees significant DDG. Determining affinities from crystal soaks is, of course, overkill. There are other easier methods that do not pose the problem you mention. However, the original post was not about determining affinities, but rather which of the two sites has the higher affinity. Is it possible that lattice constraints invert the affinity ratio? Sure. But given that the prior of the lattice DDG=0, it is more likely that the sign of the affinity ratio in crystal is the same as in solution. The likelihood of this, of course, depends on the value of solution DDG with respect to lattice DDG. Again, mutagenesis works too, but if crystals and beamtime are available, such experiment makes sense, at least in my opinion. Cheers, Ed. On Wed, 2013-11-20 at 08:20 +0100, Bärbel Blaum wrote: Hi Ed, you are right about the original question, but what I mean is this: if the occupancies (and B-factors) differ so much in crystals with IDENTICAL binding sites, i.e. identical affinities, does this not show that occupancies (and B-factors) do not reflect affinities alone, but equally local packing? There might be individual cases in which such effects can be neglected, but generally I think trying to determine affinities from crystal soaks is, hmm, not very good pratice, simply because there are other dedicated methods to do it that suffer less from side effects. Including the docking approach. Kind regards, Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: If I understand the original post correctly, the binding sites in question are not chemically identical. While it's possible that lattice may invert the order in which sites are occupied, it is not very likely given that affinity gap is sufficient to be observable by ITC. Mutagenesis is a good option too. On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: Hello, we work with proteins that have typically several chemically identical binding sites (viral capsid proteins fully assembled or as multimeric assembly-intermediates). Depending on how long at which concentrations they are soaked the chemically identical ligand pockets within one asymmetric unit are typically occupied to different levels purely because of individual crystal contacts and accessibility. I therefore think that neither soaking with different concentrations nor B-factor analysis are solid methods to determine some sort of relative affinities. I'd suggest to design mutants for either binding site and ITC measurements with the mutant proteins. This might also tell you if some sort of co-op exists between both sites. Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: IMHO, while explaining binding affinity from a structure is fun, it does not prove anything. Assuming that I understand your situation correctly, you can (relatively) easily find out from experiment which pocket has higher affinity. Just do soaks with different ligand concentrations - the expectation is that the weaker binding site will become partially occupied first. On Tue, 2013-11-19 at 04:58 +, Xiaodi Yu wrote: Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee __ Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need
Re: [ccp4bb] distinguish ligand binding sites within a protein
If I understand the original post correctly, the binding sites in question are not chemically identical. While it's possible that lattice may invert the order in which sites are occupied, it is not very likely given that affinity gap is sufficient to be observable by ITC. Mutagenesis is a good option too. On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: Hello, we work with proteins that have typically several chemically identical binding sites (viral capsid proteins fully assembled or as multimeric assembly-intermediates). Depending on how long at which concentrations they are soaked the chemically identical ligand pockets within one asymmetric unit are typically occupied to different levels purely because of individual crystal contacts and accessibility. I therefore think that neither soaking with different concentrations nor B-factor analysis are solid methods to determine some sort of relative affinities. I'd suggest to design mutants for either binding site and ITC measurements with the mutant proteins. This might also tell you if some sort of co-op exists between both sites. Baerbel Quoting Ed Pozharski epozh...@umaryland.edu: IMHO, while explaining binding affinity from a structure is fun, it does not prove anything. Assuming that I understand your situation correctly, you can (relatively) easily find out from experiment which pocket has higher affinity. Just do soaks with different ligand concentrations - the expectation is that the weaker binding site will become partially occupied first. On Tue, 2013-11-19 at 04:58 +, Xiaodi Yu wrote: Hi Wei: Based on the structure, you can calculate the binding surface between the protein and the ligand. Maybe the two binding pockets will give you two different numbers. And the larger one usually can have the higher binding affinity. You also can analyse how the ligand interacts with the protein though hydrophobic or electrostatic interaction , etc? the last, you may also compare the b factors of the ligand or the protein binding pocket regions after you refining the structure. These things may give you some hints about which binding site is more strong. Dee __ Date: Mon, 18 Nov 2013 22:45:58 -0500 From: wei.shi...@gmail.com Subject: Re: [ccp4bb] distinguish ligand binding sites within a protein To: CCP4BB@JISCMAIL.AC.UK Thank you so much for the suggestions, Tomas! Yes, my ligand is a small molecule. I have the crystal structure of the ligands bound to the protein, do I still need to computationally dock the ligand to the two pockets, can I calculate the parameters of binding directly using the crystal structure? Best, Wei On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Wei Shi, is your ligand a small molecule? If it is a small molecule, I would try to computationally dock the small molecule to two pockets separately using AutoDock, and look at the estimated free energies of binding. Best wishes, Tomas On Mon, Nov 18, 2013 at 8:55 PM, Wei Shi wei.shi...@gmail.com wrote: Hi all, I got the crystal structure of a transcription factor, and every monomer binds two molecules of the same ligand in different binding pockets. And I also did the ITC experiment, titrating the ligand into the protein, and got a U-shaped curve. The binding affinity for the first binding site is higher than the second binding site. I am wondering whether I could computationally determine from the protein-ligand complex structure that which binding site has higher affinity for the ligand and correlate the binding sites with the parameters I got from ITC experiment. Thank you so much! Best, Wei -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse / -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born
Re: [ccp4bb] Comparison of Water Positions across PDBs
http://www.ccp4.ac.uk/html/watertidy.html On 10/29/2013 04:43 PM, Elise B wrote: Hello, I am working on a project with several (separate) structures of the same protein. I would like to be able to compare the solvent molecules between the structures, and it would be best if the waters that exist in roughly the same position in each PDB share the same residue number. Basically, I want to compare solvent molecule coordinates and assign similar locations the same name in each structure. What would be the best strategy for re-numbering the water molecules such that those with similar coordinates in all the structures receive the same residue number? I'd appreciate any suggestions. Elise Blankenship -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Is there someone work with Lipids?
May I ask a question? This is a catch-22 situation. If you ask permission to ask a question, you are already asking a question. Answering this with no would probably create a wormhole. I'd also like to see a bulletin board where asking questions requires separate permission. When we prepare lipids, we always used chloroform to dissolve lipids, then resuspended in buffer. But sometime we can dissolve lipids with detergent, for example E.coli total lipids. Why we still need to use chloroform to dissolve it firstly based on standard method? Chloroform is a good way to store lipids (keep it in -80 to minimize evaporation, use teflon cap liners, warm up prior to use to avoid water absorption and top off with argon after each withdrawal). Chloroform can be easily removed (bulk with gentle stream of argon under the hood and residual by 1-2 hour dessication under oil pump) and you can then have a very good control over buffer content (charged lipids will come with counterions, of course). Thus, it's a very robust general procedure. Using detergents to dissolve lipids is much messier, I presume. Of course, this depends on what you are trying to accomplish. Cheers, Ed. -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
Re: [ccp4bb] Low 280 absorbance imidazole?
On Thu, 2013-08-22 at 10:07 -0400, Bosch, Juergen wrote: Yes, I'm also surprised why people run gradients for the capturing step ? Because we can. Joking aside, I've seen some examples where protein eluted at relatively low imidazole and upon running the gradient there remains some (minimal) overlap with non-specific binders. Mainly I just never seen consensus on what is the right imidazole concentration for the wash buffer (10mM? 50mM? may even depend on expession circumstances), running the gradient takes that uncertainty away. Also, batch method probably leaves more contamination behind. One can run imidazole gradient in 1-2 hours, batch is not really much faster, if at all. So if one has FPLC access, doing imidazole gradient seems like a good standard policy. Benefits might be minor and rare, but it is not much more work and certainly not worse than batch in any respect. My two cents (which in Russia turns into three rusty kopecks), Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] Low 280 absorbance imidazole?
On Thu, 2013-08-22 at 13:58 -0400, Bosch, Juergen wrote: well if we hit the timer after lysis, say via cell disruptor then I have my eluted protein in less than 1 hour, including 40 minutes batch binding. then proceeds to wait six weeks for crystals to appear... :) Cheers, Ed. PS. There are many ways to skin a kangaroo and ultimately indeed all things serve the Beam. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Low 280 absorbance imidazole?
On Thu, 2013-08-22 at 14:22 -0400, Bosch, Juergen wrote: But when it sits and crystallizes it is cleaner - unless some opportunistic fungal contamination helps you trim off those nasty loops that you would have omitted anyhow from your model. Jurgen, Good point and admittedly I never considered that. On the other hand, when supernatant is injected in the column on FPLC, other components that may harm the protein are either rapidly removed from the environment or immobilized on the resin and can do no further damage. Does this mean that the batch method exposes protein for longer time as you incubate it with the resin? Of course, we are splitting hairs (at least I am) - both methods work just fine most of the time and one can find problematic examples with both. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Low 280 absorbance imidazole?
According to Qiagen Since imidazole absorbs UV radiation at 280 nm, an elution profile measured at 280 nm while purifying a 6xHis tagged protein by FPLC will show an increase in absorbance above the background signal allowing quantitation of your protein. The absorbance of imidazole can vary depending on its source and purity, but elution buffer containing 250 mM imidazole usually has an A280 of 0.2–0.4. Cheers, Ed. On Wed, 2013-08-21 at 11:25 -0400, Edward A. Berry wrote: Would you happen to know what the A280 of a 1M (or .1 M) solution is? I wonder if this absorbance is really due to impurities or is the tail of a weak absorbance band of imidazole itself. I notice A280 is not one of the properties sigma lists for its imidazole. Jan wrote: Hi Bernhard, this one works for us: Sigma BioUltra imidazole, 99.5% by GC, 56749 Cheers, Jan -- Jan Abendroth Emerald BioStructures Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com On Aug 21, 2013, at 7:33 AM, Bernhard Rupp hofkristall...@gmail.com mailto:hofkristall...@gmail.com wrote: Hi Fellows, could someone please point me towards the source of a known high purity imidazole with low absorbance at 280 nm? I am facing the problem of detecting a low absorption protein in high imidazole background after IMAC gradient elution. In the UV spectra of the 2 imidazoles I checked there is some contaminant that absorbs at 280… Thx, BR Bernhard Rupp Marie Curie Incoming International Fellow Innsbruck Medical University Schöpfstrasse 41 A 6020 Innsbruck – Austria +43 (676) 571-0536 bernhard.r...@i-med.ac.at mailto:bernhard.r...@i-med.ac.at Dept. of Forensic Crystallography k.-k. Hofkristallamt Vista, CA 92084 001 (925) 209-7429 b...@ruppweb.org mailto:b...@ruppweb.org b...@hofkristallamt.org mailto:b...@hofkristallamt.org http://www.ruppweb.org/ --- -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Problems with SANS data analysis
This question may be better suited for more small-angle-oriented forum, e.g. http://www.saxier.org/forum/ On 08/07/2013 11:22 AM, Remec, Mark wrote: Dear CCP4bb, I have a few questions concerning SANS data recently collected that I'm having trouble analyzing. The data was collected at 2 different detector distances (4m, 2.5m) to achieve higher q-range, but I worry that the curves don't overlap enough at intermediate q, which might indicate a problem with the data. The links below are pictures of the corresponding datasets, before truncating the 4m high-q data and merging them into one. Is there a problem evident with the data, or am I imagining a problem? http://postimg.org/image/qb00y20qr/ http://postimg.org/image/8trbp7akj/ http://postimg.org/image/hni86axj7/ http://postimg.org/image/3sjxnu343/ http://postimg.org/image/4ysj0dgsj/ http://postimg.org/image/9ypz8bmf7/ http://postimg.org/image/m358pazb7/ http://postimg.org/image/jzuthmzib/ My second question concerns the values obtained in the analysis of the final scattering curves. The second sample in my experiment shows serious deviation in the values obtained for I(0) and Rg by Guinier analysis compared to the values obtained by the P(r) analysis. In other words, either the P(r) values match the Guinier and the P(r) fit is terrible, or else the P(r) fit is good but doesn't match the Guinier at all (5-10 difference in Rg, 2x difference in I(0)). I've checked to make sure the buffer subtraction algorithm was OK, and I'm pretty certain that the buffers were exact matches, so I don't know how to explain this variation. There's no evidence of aggregation or polydispersity to throw off the values, either. Does anyone know how this can happen? -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] mmCIF as working format?
On 08/07/2013 01:51 PM, James Stroud wrote: In the long term, the MM structure community should perhaps get its inspiration from SQL For this to work, a particular interface must monopolize access to structural data. Then maintainers of that victorious interface could change the underlying format whichever way they want while supplying the never ending stream of useful features. And all other programs would be just frontends to the interface. As long as data format remains easily readable and there is more than one person willing to fiddle with code, persistence or at the very least backward compatibility of the data format will remain a (minor to me) issue. It is also important that it is much easier to write a pdb parser in your favourite language than to implement general purpose relational database management system. For full disclosure, I personally do not share the apocalyptic feeling about transition to mmCIF. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] mmCIF as working format?
On 08/07/2013 03:54 PM, James Stroud wrote: On Aug 7, 2013, at 1:06 PM, Ed Pozharski wrote: On 08/07/2013 01:51 PM, James Stroud wrote: In the long term, the MM structure community should perhaps get its inspiration from SQL For this to work, a particular interface must monopolize access to structural data. Not necessarily, although the alternative pathway might be more idealistic and hence unrealistic. All that needs to happen is that the community agree on 1. What is the finite set of essential/useful attributes of macromolecular structural data. 2. What is the syntax of (a) accessing and (b) modifying those attributes. 3. What is the syntax of selecting subsets of structural data based on those attributes. The resulting syntax (i.e. language) itself should be terse, easy to learn, easy to use, and preferably easy to implement. If such a standard is created, then I believe awk-ing/grep-ing/sed-ing/etc PDBs and mmCIFs would quickly become historical. James James, frankly, I am not sure which part of your description is not equivalent to monopolistic interface. If I understand your proposal and reference to SQL correctly, you want some scripting language that sounds like simple English. Is the advantage over existing APIs here that one does not need to learn Python, C++, (or, heaven forbid, FORTRAN)? I.e. programs would look like this --- GRAB protein FROM FILE best_model_ever.cif; SELECT CHAIN A FROM protein AS chA; SET chA BFACTORS TO 30.0; GRAB data FROM FILE best_data_ever.cif; BIND protein TO data; REFINE protein USING BUSTER WITH TLS+ANISO; DROP protein INTO FILE better_model_yet.cif; --- Not necessarily a bad idea but now through the fog of time I remember something oddly reminiscent... ah, CNS! (for those googling for it it's not the central nervous system :). Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] mmCIF as working format?
James, On 08/07/2013 05:36 PM, James Stroud wrote: Anyone can learn Python in an hour and a half. Isn't this a bit of an exaggeration? Python is designed to be easy to learn, but we probably talking about different definitions of learning and anyone. I.e. programs would look like this --- GRAB protein FROM FILE best_model_ever.cif; SELECT CHAIN A FROM protein AS chA; SET chA BFACTORS TO 30.0; GRAB data FROM FILE best_data_ever.cif; BIND protein TO data; REFINE protein USING BUSTER WITH TLS+ANISO; DROP protein INTO FILE better_model_yet.cif; --- Not necessarily a bad idea but now through the fog of time I remember something oddly reminiscent... ah, CNS! (for those googling for it it's not the central nervous system :). Although a little too much like natural language, it is not a bad idea. But, where is the link describing the layer of CNS that looks like that? I should probably use tongue-in-cheek/tongue-in-check markup next time to prevent my poor attempt at humorous tribute to CNS from being understood so literally. At the very least you might agree that CNS is the closest thing we ever had to MX-oriented general purpose interpreter. Your quote is also from below-the-magic-line-do-not-change area of a CNS script. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] mmCIF as working format?
On 08/07/2013 05:54 PM, Nat Echols wrote: Personally, if I need to change a chain ID, I can use Coot or pdbset or many other tools. Writing code for this should only be necessary if you're processing large numbers of models, or have a spectacularly misformatted PDB file. Again, I'll repeat what I said before: if it's truly necessary to view or edit a model by hand or with custom shell scripts, this often means that the available software is deficient. PLEASE tell the developers what you need to get your job done; we can't read minds. Nat, I don't think anyone here really means that the only way to change a chain ID is to write, say, a perl script. But an interpreter of the kind advocated by James (as much as I have hijacked/misinterpreted his vision) could indeed be very useful for people pursuing simple bioinformatics projects and new ways to analyse structural models. While I understand your view that everyone should seek assistance from developers with every problem encountered, I also recall some reasonable idea about self-sufficiency that should cover scientific research (something like give man a fish and you feed him for a day, teach him to fish and he starts paying taxes... something along these lines ;). There is a difference betweens tools that allow to easily perform useful non-standard analysis and highly specialized tools that strive to cover every situation imaginable. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] ctruncate bug?
On 06/21/2013 10:19 AM, Ian Tickle wrote: If you observe the symptoms of translational NCS in the diffraction pattern (i.e. systematically weak zones of reflections) you must take it into account when calculating the averages, i.e. if you do it properly parity groups should be normalised separately (though I concede there may be a practical issue in that I'm not aware of any software that currently has this feature). Ian, I think this is exactly what I was trying to emphasize, that applying some conversion to raw intensities may have negative impact when conversion is based on incorrect or incomplete assumptions. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] ctruncate bug?
Dear Kay and Jeff, frankly, I do not see much justification for any rejection based on h-cutoff. FrenchWilson only talk about I/sigI cutoff, which also warrants further scrutiny. It probably could be argued that reflections with I/sigI-4 are still more likely to be weak than strong so F~0 seems to make more sense than rejection. The nature of these outliers should probably be resolved at the integration stage, but these really aren't that numerous. As for h-4 requirement, I don't see FrenchWilson even arguing for that anywhere in the paper. h variable does not reflect any physical quantity that would come with prior expectation of being non-negative and while the posterior of the true intensity (for acentric reflections) is distributed according to the truncated normal distribution N(sigma*h, sigma^2), I don't really see why h-4 is bad. From what I understand, Kay has removed h-cutoff from XDSCONV (or never included it in the first place). Perhaps ctruncate/phenix should change too? Or am I misunderstanding something and there is some rationale for h-4 cutoff? Cheers, Ed. On Wed, 2013-06-19 at 06:47 +0100, Kay Diederichs wrote: Hi Jeff, what I did in XDSCONV is to mitigate the numerical difficulties associated with low h (called Score in XDSCONV output) values, and I removed the h -4 cutoff. The more negative h becomes, the closer to zero is the resulting amplitude, so not applying a h cutoff makes sense (to me, anyway). XDSCONV still applies the I -3*sigma cutoff, by default. thanks, Kay -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
Re: [ccp4bb] sigma value in structure file
The only thing that seems to make sense is bonds rmsd - but you should ask the annotator for specifics directly. If it is bonds rmsd, this has been discussed many times - just google rmsd bonds ccp4bb and look for most recent entries. On Mon, 2013-06-17 at 12:11 +0530, Faisal Tarique wrote: Dear all During PDB deposition the annotator me to verify and review the sigma value in the structure file which in my case was -0.03. I have some basic queries and request you all to please answer them. My first question is, what actually is a sigma value of a structure file. 2nd) where the value is mentioned?. 3rd) and What is the optimum sigma value range ? -- Regards Faisal School of Life Sciences JNU -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
[ccp4bb] ctruncate bug?
I noticed something strange when processing a dataset with imosflm. The final output ctruncate_etc.mtz, contains IMEAN and F columns, which should be the conversion according to FrenchWilson. Problem is that IMEAN has no missing values (100% complete) while F has about 1500 missing (~97% complete)! About half of the reflections that go missing are negative, but half are positive. About 5x more negative intensities are successfully converted. Most impacted are high resolution shells with weak signal, so I am sure impact on normal refinement would be minimal. However, I am just puzzled why would ctruncate reject positive intensities (or negative for that matter - I don't see any cutoff described in the manual and the lowest I/sigI for successfully converted reflection is -18). Is this a bug or feature? Cheers, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
Re: [ccp4bb] ctruncate bug?
Jeff,
thanks - I can see the same equation and cutoff applied in ctruncate
source.Here is the relevant part of the code
// Bayesian statistics tells us to modify I/sigma by
subtracting off sigma/S
// where S is the mean intensity in the resolution shell
h = I/sigma - sigma/S;
// reject as unphysical reflections for which I -3.7 sigma,
or h -4.0
if (I/sigma -3.7 || h -4.0 ) {
nrej++;
if (debug) printf(unphys: %f %f %f %f\n,I,sigma,S,h);
return(0);
}
This seems to be arbitrary cutoff choice, given that they are
hard-coded. At the very least, cutoffs should depend on the total
number of reflections to represent famyliwise error rates.
It is however the h-based rejection that seems most problematic to me.
In the dataset in question, up to 20% reflections are rejected in the
highest resolution shell (granted, I/sigI there is 0.33). I would
expect that reflections are rejected when they are deemed to be outliers
due to reasons other than statistical errors (e.g. streaks, secondary
lattice spots in the background, etc). I must say that this was done
with extremely good quality data, so I doubt that 1 out of 5 reflections
returns some physically impossible measurement.
What is happening is that sigma=3S in the highest resolution shell,
and for many reflection h-4.0. This does not mean that reflections are
unphysical though, just that shell as a whole has mostly weak data (in
this case 89% with I/sigI2 and 73% with I/sigI1).
What is counterintuitive is why do I have to discard reflections that
are just plain weak, and not really outliers?
Cheers,
Ed.
On 06/17/2013 10:29 PM, Jeff Headd wrote:
Hi Ed,
I'm not directly familiar with the ctruncate implementation of French
and Wilson, but from the implementation that I put into Phenix (based
on the original FW paper) I can tell you that any reflection where
(I/sigI) - (sigI/mean_intensity) is less than a defined cutoff (in our
case -4.0), then it is rejected. Depending on sigI and the mean
intensity for a given shell, this can result in positive intensities
that are also rejected. Typically this will effect very small positive
intensities as you've observed.
I don't recall the mathematical justification for this and don't have
a copy of FW here at home, but I can have a look in the morning when
I get into the lab and let you know.
Jeff
On Mon, Jun 17, 2013 at 5:04 PM, Ed Pozharski epozh...@umaryland.edu
mailto:epozh...@umaryland.edu wrote:
I noticed something strange when processing a dataset with
imosflm. The
final output ctruncate_etc.mtz, contains IMEAN and F columns, which
should be the conversion according to FrenchWilson. Problem is that
IMEAN has no missing values (100% complete) while F has about 1500
missing (~97% complete)!
About half of the reflections that go missing are negative, but
half are
positive. About 5x more negative intensities are successfully
converted. Most impacted are high resolution shells with weak signal,
so I am sure impact on normal refinement would be minimal.
However, I am just puzzled why would ctruncate reject positive
intensities (or negative for that matter - I don't see any cutoff
described in the manual and the lowest I/sigI for successfully
converted
reflection is -18).
Is this a bug or feature?
Cheers,
Ed.
--
I don't know why the sacrifice thing didn't work.
Science behind it seemed so solid.
Julian, King of Lemurs
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs
Re: [ccp4bb] Concerns about statistics
On 06/14/2013 07:00 AM, John R Helliwell wrote: Alternatively, at poorer resolutions than that, you can monitor if the Cruickshank-Blow Diffraction Precision Index (DPI) improves or not as more data are steadily added to your model refinements. Dear John, unfortunately the behavior of DPIfree is less than satisfactory here - in a couple of cases I looked at it just steadily improves with resolution. Example I have in front of me right now takes resolution down from 2.0A to 1.55A, and DPIfree goes down from ~0.17A to 0.09A at almost constant pace (slows down from 0.021 A/0.1A to 0.017 A/0.1A around 1.75A). Notice that in this specific case I/sigI at 1.55A is ~0.4 and CC(1/2)~0.012 (even this non-repentant big-endian couldn't argue there is good signal there). DPIfree is essentially proportional to Rfree * d^(2.5) (this is assuming that No~1/d^3, Na and completeness do not change). To keep up with resolution changes, Rfree would have to go up ~1.9 times, and obviously that is not going to happen no matter how much weak data I throw in. The maximum-likelihood e.s.u. reported by Refmac makes more sense in this particular case as it clearly slows down big time around 1.77A (see https://plus.google.com/photos/113111298819619451614/albums/5889708830403779217). Coincidentally, Rfree also starts going up rapidly around the same resolution. If anyone is curious what's I/sigI is at the breaking point it's ~1.5 and CC(1/2)~0.6. And to bash Rmerge a little more, it's 112%. So there are two questions I am very much interested in here. a) Why is DPIfree so bad at this? Can we even believe it given it's erratic behavior in this scenario? b) I would normally set up a simple data mining project to see how common this ML_esu behavior is, but there is no easily accessible source of data processed to beyond I/sigI=2, let alone I/sigI=1 (are structural genomics folks reading this and do they maybe have such data to mine?). I can look into all of my own datasets, but that would be a biased selection of several crystal forms. Perhaps others have looked into this too, and what are your observations? Or maybe you have a dataset processed way beyond I/sigI=1 and are willing to either share it with me together with a final model or run refinement at a bunch of different resolutions and report the result (I can provide bash scripts as needed). Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Concerns about statistics
On Thu, 2013-06-13 at 08:44 -0700, Andrea Edwards wrote: In this case, the author should report a correlation coefficient along with the other standard statistics (I/sigI, Rmerg, Completeness, redundancy, ect.)? Won't hurt. What about Rpim instead of Rmerg? and if Rpim is reported, what should be the criteria for resolution cutoff? Rmerge is known to be deeply flawed for ~15 years. IMHO, it shall not be reported at all. While Rpim is better, the whole point of KarplusDiederichs is that R-type measures are not very useful in deciding resolution cutoff. Also, if this paper is the new standard how should we regard statistic reported in the literature? We should keep in mind that conservative resolution cutoff criteria has been used in the field for decades. Or.. more importantly, how do we go about reviewing current literature that does not report this statistic? Structures refined up to I/sigma=2 should be considered likely to have been refined to resolution that was cut off too low. With that said, I am pretty sure that in vast majority of cases structural conclusions derived with I/s=2 vs CC1/2=0.5 vs DR=0 cutoff will be essentially the same. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Concerns about statistics
Tim, my personal preference always was I/sigI=1. In my Scalepack days, I always noticed that ~30% of the reflections in the I/sigI=1 shells had I/sigI2, and formed an unverified belief that there should be some information there. In my experience, CC1/2=0.5 would normally yield I/sigI~1, not 2. This is based predominantly on Scala/Aimless. Cheers, Ed. On Thu, 2013-06-13 at 18:20 +0200, Tim Gruene wrote: On 06/13/2013 06:16 PM, Ed Pozharski wrote: [...] With that said, I am pretty sure that in vast majority of cases structural conclusions derived with I/s=2 vs CC1/2=0.5 vs DR=0 cutoff will be essentially the same. Hi Ed, in my experience, CC(1/2) 0.7 corresponds quite well to I/sigI 2.0 rather than CC(1/2) 0.5 (again, with the default resolution shells from xprep that also plots CC(1/2) vs. resolution. Are above numbers based on experience, too? If so, which program do you usually use to look at these statistics? Tim -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
Re: [ccp4bb] Extracting .pdb info with python
ATOM records have fixed format so you can (and should) use string slicing instead, like so (one-liner) serial, aname, altloc, resn, chid, resi, insCode, x, y, z, occ, b, element, q = line[6:11], line[12:16], line[16], line[17:20], line[21], line[22:26], line[26], line[30:38], line[38:46], line[46:54], line[54:60], line[60:66], line[76:78], line[78:80] or with more explicit typing serial, aname, altloc, resn, chid, resi, insCode, x, y, z, occ, b, element, q = int(line[6:11]), line[12:16].strip(), line[16].strip(), line[17:20].strip(), line[21], int(line[22:26]), line[26], float(line[30:38]), float(line[38:46]), float(line[46:54]), float(line[54:60]), float(line[60:66]), line[76:78].strip(), line[78:80] Cheers, Ed. On Thu, 2013-06-06 at 04:37 +, GRANT MILLS wrote: Dear CCP4BB, I'm trying to write a simple python script to retrieve and manipulate PDB data using the following code: #for line in open(PDBfile.pdb): #if ATOM in line: #column=line.split() #c4=column[4] and then writing to a new document with: #with open(selection.pdb, a) as myfile: #myfile.write(c4+\n) Except for if the PDB contains columns which run together such as the occupancy and B-factor in the following: ATOM608 SG CYS A 47 12.866 -28.741 -1.611 1.00201.10 S ATOM609 OXT CYS A 47 14.622 -24.151 -1.842 1.00100.24 O My script seems to miscount the columns and read the two as one column, does anyone know how to avoid this? (PS, I've googled this like crazy but I either don't understand or the link is irrelevant) Any advice would help. Thanks for your time, Grant -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Extracting .pdb info with python
On Thu, 2013-06-06 at 14:41 +1000, Nat Echols wrote: You should resist the temptation to write your own PDB parser; that way lies pain and suffering. There are multiple free libraries for Python that can be used for this task - I recommend either CCTBX or BioPython (probably the latter if you don't need to do very much with the models). Well, that depends on what one is trying to accomplish. Say, I just want to count how many atoms I have that are partially occupied in an average PDB file. All I need to know is that occupancy is stored in columns 55:60 as Real(6.2), and that atom record lines begin with ATOM |HETATM. With basic python/c/perl/whatever knowledge I can write my own occupancy parser faster than this post. Getting BioPython or CCTBX to work will definitely take longer. By writing primitive parsers one also gains speed and portability, as well as extends programming skills. Basically this choice depends on the complexity of the task and long/short-term goals. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] use only companies that you know to purchase chemicals
On Wed, 2013-05-29 at 12:30 -0400, Jeremy Stevenson wrote: In this particular case you can see the website was registered in September of 2012, which is a good indication that it was set up just to scam people. Just curious - why registration date indicates unsavory nature of Jieke? -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] To build ssDNA to base pair with the other strand of DNA in coot
I am wondering whether there is a function I could use to build the missing bases in one strand of the DNA to base pair with the other strand of DNA which is complete... Calculate-Other modeling tools- Base pair... -- Coot verendus est
Re: [ccp4bb] Short contact between symmetry equivalents
It is probably a wrong question to ask here. Pretty much everything is tolerated by PDB during deposition, the report you get is an advice, not instruction. I wonder whether anyone has an example of the RCSB/PDBe/PDBj ever turning down submitted structure. The right question is whether the short contact you mention is tolerated by laws of nature. It's fairly common to have, say, a water molecule split in two positions near symmetry axis - as long as you have it at occupancy1.0, it's ok. On 05/27/2013 05:21 AM, Kavyashree Manjunath wrote: Dear users, Is short contact (1.83Ang) between an atom and symmetry equivalent of itself tolerated during deposition? I am not able to get rid of this short contact appearing after refinement. Thank you Regards Kavya -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Short contact between symmetry equivalents
On 05/27/2013 11:27 AM, ka...@ssl.serc.iisc.in wrote: Sir, Ok. It is an acetate ion which interacts with its symmetry equivalent ion only one of its oxygen atoms is closer to its symmetry equivalent and not the entire ion. So do I need to give lower occupancy for this ion? Thank you Regards Kavya It does not interact - you cannot have 1.8A distance between atoms. Assuming that it is indeed acetate it must be partially occupied, 0.5 or less. Keep in mind that when you lower occupancy you may see additional density for whatever occupies the space on the other side of the symmetry element (e.g. water) which you may need to model. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Short contact between symmetry equivalents
On 05/27/2013 12:14 PM, Kavyashree Manjunath wrote: Later I tried with 0.8, 0.99 for which the map was normal and also validation did not report it as short contact. Is it ok if I give 0.99 occupancy? Validation most likely will not report any short contacts if occupancy is 1. If the distance between atoms is still ~1.8A, you have a problem. Perhaps it is not an acetate. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] cryo condition
Matt, with this technique, how do you prevent crystal from drying up (other than doing it fast)? I know Thorne's group does this trick under oil. If you take no extra precautions, do you have an estimate of how often diffraction is destroyed by this? On the other hand, it's quite possible that what destroys resolution when crystals dry up is increase in concentration of non-volatile mother liquor components, which shouldn't be happening here to the same degree. Cheers, Ed. On Thu, 2013-05-23 at 14:38 +0200, Matthew BOWLER wrote: Hi Faisal, if your solvent channels are smaller than 40A in the largest dimension (most are) you can use a mesh loop to pick up the crystal and then wick away all of the mother liquor. You can then flash cool your crystal without having to transfer the crystal to another solution. Good luck, Matt -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] DNA version converter
Tim, naturally, the issue is specific to a particular pdb file (it would be indeed strange if the conversion tool from a major software package would fail in general sense). Ed. On Wed, 2013-05-22 at 08:12 +0200, Tim Gruene wrote: Hi Ed, # pdbvconv -p udo.pdb -o udov.pdb # pdbvconv -p udov.pdb -o udovv.pdb # diff -q udo.pdb udov.pdb Files udo.pdb and udov.pdb differ # diff -q udov.pdb udovv.pdb Files udov.pdb and udovv.pdb differ # diff -q udo.pdb udovv.pdb # it works for me, Version: 1.0 (11 June 2008) from buster 2.10. Which version of pdbvconv are you using? Best, Tim On 05/21/2013 10:40 PM, Ed Pozharski wrote: On 05/21/2013 04:35 PM, Francis Reyes wrote: Since you're using buster, have you tried global phasing's own pdbvconv tool? Naturally, but it leaves file unchanged. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] DNA version converter
On Wed, 2013-05-22 at 18:09 +0200, Tim Gruene wrote: the answers you received were correct with respect to the question you asked. If they are not satisfactory, you have not given sufficient information. Tim, Not sure when I expressed any dissatisfaction with replies I received. I asked whether someone had a simple tool to turn v3 DNA records back to v2. I got an excellent off-list suggestion to use Remediator tool from kinemage/molprobity (writen by Jeff Headd and Robert Immormino). It was probably easy to guess that I tried pdbvconv and ran into problems (in fact, buster takes care of conversion internally unless it fails). I appreciate your initial comment that pdbvconv works for you and was simply pointing out that my observations are not of general nature and obviously specific to a particular file I was trying to convert. It appears that you were offended by that and if so, it was not my intent. In my defense, I only asked for available conversion tools and did not ask specifically for help with pdbvconv. With this in mind, I hope I can be forgiven for not posting unpublished structural model on a bulletin board in order to provide sufficient information for answering a question I did not ask. If you are interested in specifics, the problem was that some other program made residue names left-justified. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] DNA version converter
Dear Tim, I am sorry that you feel offended. It is rather unfortunate that you got an impression from my secondary comment that I am asking for help with pdbvconv when I wasn't. It is also rather unfortunate that I have figured out what the problem was myself and therefore did not have an opportunity to ask for and fully utilize your help and that of other ccp4bb members. In general, it is rather unfortunate that helping others often feels and occasionally is a waste of time. Hopefully this unfortunate experience will not dissuade you from positively contributing to the ccp4bb to the same extent to which it dissuades me from ever again soliciting any advice through this venue. Cheers, Ed. PS. Naturally, at the time of the original post and the subsequent one you responded to I did not yet know what was wrong with the specific pdb file. To have such knowledge and yet ask why pdbvconv fails (which I did not ask) would be both stupid and evil. Then again, I may be both of these things. On Wed, 2013-05-22 at 21:55 +0200, Tim Gruene wrote: Dear Ed, I do feel offended because I follow the ccp4bb with the intention of helping people with my answers, which does take time. If it turns out the I wasted my time because of the lack of information I consider the question asked not follow what I consider netiquette. I doubt that one line from a PDB file or the point that you have used pbdvconv before (especially in case you were aware of the problem being due to a simple shift - of course I do not know whether you were aware of it when opening the thread) and it failed would have revealed any information that would give your competitors any advantage, but it would have narrowed down the problem and probably the number of people spending time trying to find and give a helpful answer. Regards, Tim On 05/22/2013 07:55 PM, Ed Pozharski wrote: On Wed, 2013-05-22 at 18:09 +0200, Tim Gruene wrote: the answers you received were correct with respect to the question you asked. If they are not satisfactory, you have not given sufficient information. Tim, Not sure when I expressed any dissatisfaction with replies I received. I asked whether someone had a simple tool to turn v3 DNA records back to v2. I got an excellent off-list suggestion to use Remediator tool from kinemage/molprobity (writen by Jeff Headd and Robert Immormino). It was probably easy to guess that I tried pdbvconv and ran into problems (in fact, buster takes care of conversion internally unless it fails). I appreciate your initial comment that pdbvconv works for you and was simply pointing out that my observations are not of general nature and obviously specific to a particular file I was trying to convert. It appears that you were offended by that and if so, it was not my intent. In my defense, I only asked for available conversion tools and did not ask specifically for help with pdbvconv. With this in mind, I hope I can be forgiven for not posting unpublished structural model on a bulletin board in order to provide sufficient information for answering a question I did not ask. If you are interested in specifics, the problem was that some other program made residue names left-justified. Cheers, Ed. -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
[ccp4bb] DNA version converter
Does anyone have a script to convert pdb file with DNA atom records from v3 back to v2? I can certainly right my own and asking only if you already have it written. Strictly speaking, this is not ccp4-related - apparently, buster expects the old format. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] DNA version converter
On 05/21/2013 04:35 PM, Francis Reyes wrote: Since you're using buster, have you tried global phasing's own pdbvconv tool? Naturally, but it leaves file unchanged. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] question about CCP4 scripts
At this point you do have the scalepack2mtz output file (BTW,
imosflm/aimless is wholeheartedly recommended by this convert), and you
can easily extract all the info from there. There is mtzdmp, of course,
but it's easier to parse the actual mtz file (hey, the records are
actually text). Like so:
egrep -oa SYMINF.{74} foo.mtz | awk '{print symm $5}'
Gives you the space group *number*, most ccp4 programs I know accept
that in addition to string symbol. But if you really need the latter,
echo symm $(egrep -oa SYMINF.{74} foo.mtz | cut -d' -f 2 | sed
s///g)
will do.
As for unit cell parameters, this should work
egrep -oa CELL.{76} foo.mtz | sed -n 1p
Keep in mind that this extracts the global cell and will be
problematic if you have multi-dataset file (which I presume you don't).
If you need Nth dataset grab DCELL record, e.g. for the dataset #1
egrep -oa DCELL.{75} foo.mtz | sed -n 2p
Cheers and
https://xkcd.com/208/
Ed
On Wed, 2013-05-08 at 23:37 -0400, Joe Chen wrote:
Hi All,
I am trying to write a shell script to streamline a few steps, one of
which is Unique, see below. As you can see, this program requires
symmetry and cell parameters. In CCP4 GUI Scalepack2mtz, these info
are automatically extracted from .sca file (first two lines). But I
don't know if there is a way to do this in script, so I don't need to
type these values for each dataset. Thank you in advance for your
help.
#!/bin/sh
# unique.exam
#
# runnnable test script for the program unique - this will use this
# program to generate a reflection list containing null values.
#
set -e
unique hklout ${CCP4_SCR}/unique_out.mtz eof
labout F=F SIGF=SIGF
symmetry p43212
resolution 1.6
cell 78.1 78.1 55.2 90.0 90.0 90.0
eof
--
Best regards,
Joe
--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /
Re: [ccp4bb] Program or server to predict Kd from complex structure
Don't believe such program/server does exist. Notice that you are asking for something that *can* predict Kd. One can *try* making such predictions and they may even be routinely in the ballpark, assuming that you are satisfied with being routinely off by, say, an order of magnitude. One can easily predict general trends. For example, larger buried apolar surface will generally result in lower Kd. As for individual Kd prediction accuracy, that's another story. It's unknown to me what your goal is, but if you are trying to replace experimental Kd determination with a magic program, please don't. Cheers, Ed. Original message From: Wei Liu we...@me.com Date: 04/18/2013 4:39 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Program or server to predict Kd from complex structure Dear all, Does anyone know a program or web server that can predict Kd value between two proteins from a solved complex structure? Regards Wei
Re: [ccp4bb] salt or not?
Protein-DNA complex crystal with channels too small for the dye is *extremely* unlikely, imho. Original message From: Ulrike Demmer ulrike.dem...@biophys.mpg.de Date: 04/15/2013 8:48 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] salt or not? Dear Careina, altough your crystals does't take up the Izit dye it sounds promising. The uptake of Izit depends on the solvent channels of the protein molecule - sometimes the dye just can't enter the molecule. Concerning the Calciumchloride - if the concentration is not too high and without other ingredients which could cause less solube salt there is the possiblity that you have got protein crystals. I once had a condition whith 34 % MPD + 0.1M buffer + 0.1 M Calciumchloride which produced nicely diffracting crystals To speak from my experience I think 1 month after setting up the trays the drops should not be dried out. If the wells are sealed properly new crytals can appear even after 1 year. You should definately check the diffraction then you will know for sure. Cheers, Ulrike
Re: [ccp4bb] DNA structures superimpose
Doesn't lsqkab work? It just needs proper atom matching. Coot definitely does superimpose DNA. One problem is that simple-lsq works on a single chain, but you can trick it by changing chain id and renumbering the other strand. As for helix rotation, you can derive it from rotation reported by lsq. But it is more common to look at DNA geometry directly for changes (3DNA or Curves+). Original message From: Veerendra Kumar (Dr) veeren...@ntu.edu.sg Date: 04/12/2013 2:18 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA structures superimpose Dear CCP4 members, Is there any program to superimpose the DNA structures? I also want to measure the relative domain rotation angle. I tried using DynDom but it does not work for me. Can someone suggest a program which can output the rotation angles? Thank you Best Regards Veerendra kumar CONFIDENTIALITY:This email is intended solely for the person(s) named and may be confidential and/or privileged.If you are not the intended recipient,please delete it,notify us and do not copy,use,or disclose its content. Towards A Sustainable Earth:Print Only When Necessary.Thank you.
Re: [ccp4bb] CCP4 Update victim of own success
On 04/12/2013 06:03 PM, Nat Echols wrote: On Fri, Apr 12, 2013 at 2:45 PM, Boaz Shaanan bshaa...@exchange.bgu.ac.il mailto:bshaa...@exchange.bgu.ac.il wrote: Whichever way the input file for the run is prepared (via GUI or command line), anybody who doesn't inspect the log file at the end of the run is doomed and bound to commit senseless errors. I was taught a long time ago that computers always do what you told them to do and not what you think you told them, which is why inspecting the log file helps. I agree in principle - I would not advocate that anyone (*especially* novices) run crystallography software as a black box. However, whether or not a program constitutes a black box has nothing to do whether it runs in a GUI or not. The one advantage a GUI has is the ability to convey inherently graphical information (plots, etc.). That it is still necessary to inspect the log file(s) carefully reflects the design of the underlying programs; ideally any and all essential feedback should also be displayed in the GUI (if one exists). Obviously there is still much work to be done here. -Nat It is hard to blame novices for running crystallography software as a black box when the websites from which they download the said software use the word automated to describe it. Because, at least according to wikipedia (another great resource that should be used with care), automation is the operation of machinery without human supervision. Checking the log-files or messages supplied by GUI seems to fall under human supervision, which automated programmes should not really require. I am not advocating return to the stone age when naming a tutorial for a widely used model building software ... for morons was probably considered a joke (not a good one too). I am just saying that it is perhaps quite predictable that with promise of automation comes the expectation of, well, automation. Whether the true automation of crystallographic structure determination may become available in the future is perhaps debatable. Whether it is already available probably isn't. On a broader question of GUI versus command line, both obviously have their uses. Mastering command line gives one flexibility and perhaps greater insight into what programmes actually do. Do I prefer a little button that opens a file chooser dialog over sam-atom-in? Absolutely. But I am glad that --pdb and --auto command line options are supplied, because I can then write a little bash pipeline to pass 50 expected protein-ligand complex datasets through simple refmac-coot cycle to quickly see which ones are interesting. In that regard, both ccp4 and phenix are doing it the right way - gui is simply a gateway to the command-line controlled code. I can then choose the interface that fits particular situation. As for the relatively new CCP4 update feature, it is absolutely awesome. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Building ideal B DNA model in Coot
On 04/04/2013 04:51 PM, 李翔 wrote: Hi everyone, I met a problem when trying to build ideal DNA model in Coot. The calculated DNA looks less than 10.5 bp/ turn, probably is about 10 bp/ turn. Is there a way for me to change the pitch to make it 10.5 bp/turn in Coot? Thanks for your kind help! Sincerely, Frank I do not know the answer to your question (but suspect the answer is no). If you want a better control over DNA conformation, consider 3DNA (http://x3dna.org) - it can build the DNA molecule from set of parameters you provide. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] COOT usage for structure comparison
On 04/04/2013 04:29 AM, Tim Gruene wrote: Dear --, Are we, the ccp4bb community, recently on the hunt to find new and exciting ways to make sure people stop asking questions? Gentleman from Moscow has clearly disclosed his full name and affiliation, but perhaps I am wrong and subtle criticism of his signature-formatting skills is highly relevant. Feel free to call me Julian from now on :) Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Rfree reflections
As I recall, number of reflections set aside for cross-validation also affects stability of sigmaA estimates. With 500 reflections and 20 resolution shells you are down to 25 reflections per shell, which may be a bit too low. Original message From: Robbie Joosten robbie_joos...@hotmail.com Date: To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rfree reflections Hi Tim, The derivation of sigma(Rw-free) is in this paper: Acta Cryst. (2000). D56, 442-450. Tickle et al. Note the difference between the sigma of weighted/generalized/Hamilton R-free and that of the 'regular' R-free (there is a 2 there somewhere). From my own tests (10 fold cross-validation on 38 small datasets) I also find sigma(R-free) = R-free/sqrt(Ntest). For large datasets you really do not need to do k-fold cross validation, because sigma(R-free) can be predicted quite well. We just need to realize that it exists, Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 26, 2013 11:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rfree reflections Hi Robbie, thank you for the explanation. Heinz Gut and Michael Hadders pointed me at Axel Brunger's publication Methods Enzymol. 1997;277:366-96., http://www.ncbi.nlm.nih.gov/pubmed/18488318, which is where I got the notion of 500-1000 from. In this article a decrease of the error margin of Rfree with n^(1/2) is mentioned (p.384), but only as an observation. Is your statement inverse proportional with the number of reflections based on some statistical treatment, or also just on observation? It is a pity that k-cross validation is not standard routine because it seems so easy and so quickly to do with nowadays computers and a simple script. But that's probably like reminding people of not using R_int anymore in favour of R_meas... Cheers, Tim On Tue, Mar 26, 2013 at 10:24:51AM +0100, Robbie Joosten wrote: Hi Tim, I don't think the 5-10% or 500-1000 reflections are real rules, but rather practical choices. The error margin in R-free is inverse proportional with the number of reflections in your test set and also proportional with R-free itself. So for R-free to be 'significant' you need some absolute number of reflections to reach your cut-off of significance. This is where the 1000 comes from (500 is really pushing the limit). You want to make sure the error margin in R and R-free are not too far apart and you probably also want to keep the test set representative of the whole data set (this is particularly important because we use hold-out validation, you only get one shot at validating). This is where the 5%-10% comes from. Another consideration for going for the 5%-10% thing is that this makes it feasible to do 'full' (i.e. k-fold) cross-validation: you only have to do 20-10 refinements. If you would go for 1000 reflections you would have to do 48 refinements for the average dataset. Personally, I take 5% and increase this percentage to maximum 10% if using 5% gives me a test set smaller than 1000 reflections. HTH, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Tuesday, March 26, 2013 09:33 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Rfree reflections Dear all, I recall that the set of Rfree reflections should be 500-1000, rather than 5- 10%, but I cannot find the reference for it (maybe Ian Tickle?). I would therefore like to be confirmed or corrected: Is there an absolute number required for Rfree to be significant, i.e. 500-1000 irrespective of the total number of unique reflections in the data set, or is it 5-10% (as a compromise)? Thanks and regards, Tim -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Isothermal titration calorimetry
This might have changed, but in the past file formats were different. Microcal files are text, while TA's are binary. I do have the actual description of TA's format if anyone is interested, but it must be easier to use native text export than write a converter. Original message From: Bosch, Juergen jubo...@jhsph.edu Date: To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry Hi George, this is probably a very stupid suggestion and you likely have tried it, but I'll suggest the obvious nevertheless. What happens to your .nitc file when you rename it to .itc can you read it in Origin then ? Jürgen On Mar 24, 2013, at 6:39 AM, George Kontopidis wrote: Chris, indeed nanoITC instrument analysis software is very robust and user friendly (probable more friendly than microcal, GE). Although when you need to subtract Q (heat) values (from 2 or 3 blank experiments) from your experimental data you cannot. NanoITC software can subtract Q values only from 1 blank experiment. Also if you want to present your data in a form of heat/mol in Y (vertical) axes again you cannot. It presents data in Y axes only in form of heat/injection. If you have found a way to extract 2 or 3 blank experiments from experimental data or present data in form of heat/mol, please let me know it will be very useful. The main problem in the output files from nanoITC come with an extension .nitc, by default. Unfortunately Origin (that can do all the above) can read only, filenames with an extension .itc Cheers, George -Original Message- From: Colbert, Christopher [mailto:christopher.colb...@ndsu.edu] Sent: Saturday, March 23, 2013 5:56 PM To: George Kontopidis; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry George, would you please explain your comments? We've found the TA Instruments analysis software very robust and user friendly. We have the low volume nanoITC from TA instruments and get equivalent #'s in our comparison tests to the Microcal instrument. Cheers, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 On 3/23/13 8:47 AM, George Kontopidis gkontopi...@vet.uth.gr wrote: Keep in mind that output files from nanoITC, TA instrument cannot be red by Origin. At some point you will need to analyse your data further. George -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Saturday, March 23, 2013 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry It might be worth to consider the question more in detail. Do you want to study thermodynamics of the interaction, or a KD would do? If the former, you need ITC. If the latter, and you want to study things at the level of KD only, maybe investing on a plate reader, thermophoresis, or some biosensor technology (spr or interferometry based systems) should be considered. Then, what interactions will you study with the ITC? In general, I would agree that the lower sample volume is worth the nano options, but depending on the typical systems under study, sometimes the gain on sample quantity is not worth the money - while many times its worth it. John is if course right that for studying specific systems as the one he describes the 200 is great. A. Sent from my iPhone On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.com wrote: I would recommend the Microcal ITC 200, hands down. Not only is it an amazing instrument with the optional automated sample loader (which is worth every penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 binding to a weak cognate protein. I believe this was the first time ITC was ever used with full length p53, as it is so labile and just loves immediately to oligomerize. Sample sizes pay for the instrument. Best, John John Fisher, M.D./PhD St. Jude Children's Research Hospital Department of Oncology Department of Structural Biology W: 901-595-6193 C: 901-409-5699 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.com wrote: Dear All, I am sorry for the off topic question. I am going to buy ITC to study protein-protein protein-ligand interactions I am comparing microcal, GE and nanoITC, TA instrument.. any suggestions, recommendations, good experiences or bad experiences. is there a better system. Thank in advance for the help. Regards Sameh -- Sameh Soror Postdoc. fellow .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926
Re: [ccp4bb] How to convert file format from CNS to CCP4
On 03/23/2013 09:59 AM, Wei Feng wrote: Can you help me to check out why these maps can not be converted by sftools? sftools is not for manipulating map files. Mapman from uppsala software factory would be a good choice. xdlmapman, a gui frontend to it, used to be part of ccp4. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Philosophical question
On 03/19/2013 02:41 PM, Jacob Keller wrote: I don't understand this argument, as it would apply equally to all features of the theoretical LUCA No it won't. Different features would have different tolerance levels to modifications. Philosophically, one is wrong to expect that living organisms will evolve in a fashion that we find optimal. Whenever I feel that a protein behaves in a way I find stupid, I simply say giraffe laryngeal nerve and all comes back to normal.
Re: [ccp4bb] Philosophical question
Jacob, So you'd have to explain why the codon convention is so intolerant/invariant relative to the other features--it seems to me that either it is at an optimum or there is some big barrier holding it in place. Because altering codon convention will result in massive translation errors. However the original question refers to start codon and its relation to methionine. Notice that AUG is the *only* codon for methionine. If you change amino acid specificity of the methionine tRNA synthetase, you'd replace every methionine in every protein. It is very unlikely that an organism other than one with a very small genome can survive that. Given high fidelity required of tRNA synthetases, changing their specificity is also not easy. Most mutations are likely to incapacitate the enzyme rather than switch its specificity, resulting in organism that is unable to develop (due to stalled translation), let alone survive. As for the optimization part - I am also not sure what significant benefit you expect from replacing starting methionine with a different amino acid. It is mostly removed anyway. Why that is? My (uneducated) guess is that it is rarely structural and there is benefit in recycling it. Cheers, Ed.
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Check for translational NCS And you are way too conservative with resolution. Even those holding onto the Rmerge-dictated past would probably acquiesce to lower I/sig cutoff. If you are using aimless, follow its recommendations based on CC1/2, it's good for you. Cheers, Ed. On Fri, 2013-03-15 at 15:39 -0300, Andrey Nascimento wrote: Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] statistical or systematic? bias or noise?
Pete, Actually, I was trying to say the opposite - that the decision to include something in the model (or not) could change the nature of the error. Duly noted Pete PS - IIUC := ? IIUC - If I Understand Correctly -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
Re: [ccp4bb] [ccp4b] statistical or systematic? bias or noise?
Adam, OK, seems like you are going with it's always statistical error we just don't yet know what it is option. Ed. On Tue, 2013-03-12 at 16:15 +, Adam Ralph wrote: Hi Ed, You can have both types of error in a single experiment, however you cannot determine statistical (precision or as Ian says uncontrollable) error with one experiment. The manufacturer will usually give some specs on the pipette, 6ul +/- 1ul. In order to verify the specs you would need to perform many pipetting experiments. But even if the manufacturer does not give any specs you still know that the pipette is not perfect and there will be a statistical error, you just do not know what it is. In theory, accuracy or bias could be determined with one experiment. Lets say you thought you had a 6ul pipette but actually it was a 12ul pipette. If you then compare the 'new' pipette against a standard you could tell if it was inaccurate. Of course normally you would repeat this experiment as well because of statistical error. If detected bias can be removed. Systematic error may not be so easily detected. What if the standard is also biased. Adam One can say it's inaccuracy when it is not estimated and imprecision when it is. Or one can accept Ian's suggestion and notice that there is no fundamental difference between things you can control and things you can potentially control. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] statistical or systematic? bias or noise?
Kay, the latter is _not_ a systematic error; rather, you are sampling (once!) a statistical error component. OK. Other words, what is potentially removable error is always statistical error, whether it is sampled or not. So is it fair to say that if there are some factors that I either do not know about, willfully choose to ignore or just cannot sample, then I am underestimating precision of the experiment? Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] statistical or systematic? bias or noise?
Ian, thanks - I think I had it backwards after reading your first post and thought of controllable errors being those that can be brought under conrtol by sampling, whereas uncontrollable would be those that cannot be sampled and therefore their amplitude is unknown. Yet you also seem to agree that characterization is dependent on specifics of experimental setup, leaving the door open for the possibility that noise-vs-bias choice may be driven by experimental circumstance. And in practice, wouldn't it be more consistent to stick with the definition that statistical error/noise/precision is defined by what is really sampled? Because if some factor is not sampled, I have zero knowledge of the corresponding error magnitude. I agree with Tim that not sampling what can be easily sampled is a poorly designed experiment, but it can also be characterized (which is probably a nicer term) as an experiment with large systematic error (due to poor design). Cheers, Ed. On Wed, 2013-03-13 at 12:33 +, Ian Tickle wrote: Ed, sorry for delay. I was not trying to make any significant distinction between controllable and potentially controllable: from a statistical POV they are the same thing. The distinction is purely one of practicality, i.e. within the current experimental parameters is it possible to eliminate the systematic error, for example is there a calibration step where you determine the systematic error by use of a standard of known concentration. The error is still controllable regardless of whether you actually take the trouble to control it! Note that the experimental setup has not changed, you are merely using the same apparatus in a different way but any random errors associated with the measurements will still be present. Of course if you change the experimental setup (note that this potentially includes the experimenter!) then all bets are off! It's very important to describe the experimental setup precisely before you attempt to characterise the errors associated with a particular setup. BTW I agree completely with Kay's analysis of the problem: as he said you are sampling (once!) a statistical error component. This is what I was trying to say, he just said it in a much more concise way! This random (uncontrollable) error then gets propagated through the sequence of steps in the experiment along with all the other uncontrollable errors. Cheers -- Ian On 11 March 2013 19:04, Ed Pozharski epozh...@umaryland.edu wrote: Ian, thanks for the quick suggestion. On Mon, 2013-03-11 at 18:34 +, Ian Tickle wrote: Personally I tend to avoid the systematic vs random error distinction and think instead in terms of controllable and uncontrollable errors: systematic errors are potentially under your control (given a particular experimental setup), whereas random errors aren't. Should you make a distinction then between controllable (cycling cuvette in and out of the holder) and potentially controllable errors (dilution)? And the latter may then become controllable with a different experimental setup? Cheers, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] statistical or systematic? bias or noise?
OK. Other words, what is potentially removable error is always statistical error, whether it is sampled or not. Clarification - what I meant is potentially removable by proper sampling and reducing standard error to zero with infinite number of measurements. Not removable by better calibration or experimental setup. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
Re: [ccp4bb] statistical or systematic? bias or noise?
Ian, On Wed, 2013-03-13 at 19:46 +, Ian Tickle wrote: So I don't see there's a question of wilfully choosing to ignore. or not sampling certain factors: if the experiment is properly calibrated to get the SD estimate you can't ignore it. So perhaps I can explain better by using the same example of protein concentration measurement. It is certainly true that only taking one dilution is poor design. (Although in crystallization practice it may not matter given that it is not imperative to have a protein exactly at 10 mg/ml, 9.7 will do). If I don't bother including pipetting precision in my error estimate either by direct experiment or by using manufacturer's declaration I am willfully ignoring this source of error. That would be wrong. But what if I only have one measurement worth of sample? And pipetting precision cannot be calibrated (I know it can be so this is hypothetical - say pipettor was stolen and company that made it is out of business, their offices burned down by raging mob). Is the pipetting error now systematic because experimental situation (not design) prevents it from being sampled or estimated? I actually like the immutable error type better for my own purposes, but I am trying to see whether some argument might stand that allows some error that can be sampled to be called inaccuracy nonetheless. Cheers and thanks, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
[ccp4bb] qtrview command line options
Is there some way of opening a log file (specifically, the pointandscale.log that imosflm bridge to scala generates) with qtrview from command line? I tried, of course, this qtrview pointandscale.log but it opens empty, no log-file. I tried qtrview -h and qtrview --help and man qtrview but there is seemingly no documentation. I found the source code (yes, I can google) and can deduce that available options at startup are --log-file --report-xml --report-xrt --inp-file The only thing that works is qrtview --log-file pointandscale.log but that only shows me the log-file itself, i.e. no graphs etc. I understand that the program was designed primarily for ccp4 gui and I know loggraph (and it works). By the way, checkout instructions for the qtrview repository at https://fg.oisin.rc-harwell.ac.uk/projects/qtrview/ don't work throwing this error bzr: ERROR: Connection error: curl connection error (server certificate verification failed. CAfile: /etc/ssl/certs/ca-certificates.crt CRLfile: none) on https://fg.oisin.rc-harwell.ac.uk/anonscm/bzr/qtrview/.bzr/smart Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[ccp4bb] statistical or systematic? bias or noise?
Salve, I would like to solicit opinions on a certain question about the relationship between statistical and systematic error. Please read and consider the following in its entirety before commenting. Statistical error (experiment precision) is determined by the degree to which experimental measurement is reproducible. It is derived from variance of the data when an experiment is repeated multiple times under otherwise identical conditions. Statistical error is by its very nature irremovable and originates from various sources of random noise, which can be reduced but not entirely eliminated. Systematic error (experiment accuracy) reflects degree to which precise average deviates from a true value. Theoretically, corrections can be introduced to the experimental method that eliminate various sources of bias. Systematic error refers to some disconnect between the quantities one tries to determine and what is actually measured. The issue is whether the classification of various sources of error into the two types depends on procedure. Let me explain using an example. To determine the concentration of a protein stock, I derive extinction coefficient from its sequence, dilute it 20x to and take OD measurement. The OD value is then divided by extinction coefficient and inflated 20 times to calculate concentration. So what is the statistical error of this when I am at the spectrophotometer? I can cycle sample cuvette in and out of the holder to correct for reproducibility of its position and instrument noise. This gives me the estimated statistical error of the OD measurement. Scaled by extinction coefficient and dilution factor, this number corresponds to the statistical error (precision) of the protein concentration. There are two sources of the systematic error originating from the two factors used to convert OD to concentration. First is irremovable inaccuracy of the extinction coefficient. Second: dilution factor. Here main contribution to the systematic error is pipetting. Importantly, this includes both systematic (pipettor calibration) and statistical (pipetting precision) error. Notice that I only prepared one sample, so if on that particular instance I picked up 4.8ul and not 5.0ul, this will translate into systematically underestimating protein concentration, even though it could have equally likely been 5.2ul. So if pipetting error could have contributed ~4% into the overall systematic error while the spectrophotometer measures with 0.1% precision, it makes sense to consider how this systematic error can be eliminated. The experiment can be modified to include multiple samples prepared for OD determination from the same protein stock. An interesting thing happens when I do that. What used to be a systematic error of pipetting now becomes statistical error, because my experiment now includes reproducing dilution of the stock. In a nutshell, Whether a particular source of error contributes to accuracy or precision of an experiment depends on how experiment is conducted. And one more thing. No need to waste precious protein on evaluating error of pipetting. I can determine that from a separate calibration experiment using lysozyme solution of comparable concentration/surface tension. Technically, a single measurement has accuracy of said 4% (padded by whatever is error in extinction coefficient). But one can also project that with actual dilution repeats, the precision would be this same 4% (assuming that this is a dominant source of error). So, is there anything wrong with this? Naturally, the question really is not about extinction coefficients, but rather about semantics of what is accuracy and what is precision and whether certain source of experimental error is rigidly assigned to one of the two categories. There is, of course, the wikipedia article on accuracy vs precision, and section 3.1 from Ian's paper (ActaD 68:454) can be used as a point of reference. Cheers, Ed. -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the great Tao is abandoned, Ideas of humanitarianism and righteousness appear. When intellectualism arises It is accompanied by great hypocrisy. When there is strife within a family Ideas of brotherly love appear. When nation is plunged into chaos Politicians become patriotic. -- / Lao Tse /
Re: [ccp4bb] statistical or systematic? bias or noise?
Tim, On Mon, 2013-03-11 at 18:51 +0100, Tim Gruene wrote: I don't share your opinion about a single measurement translating into a systematic error. I would call it a poorly designed experiment in case you were actually iterested in how accurately you determined the protein concentration. OK. As I said, this is not about protein concentration, but let's say I only have about 6ul of protein sample, so that I can only have *one* dilution. Would pipetting uncertainty then be considered systematic error or statistical error? I am afraid this is a matter of unsettled definitions. By the way, it wasn't an opinion, more of an option in interpretation. I can say that whatever is not sampled in a particular experimental setup is systematic error. Or I can say that (as you seem to suggest, and I like this option better) that whenever there is a theoretical possibility of sampling something, it is statistical error even though the particular setup does not allow accounting for it. Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] statistical or systematic? bias or noise?
Ian, thanks for the quick suggestion. On Mon, 2013-03-11 at 18:34 +, Ian Tickle wrote: Personally I tend to avoid the systematic vs random error distinction and think instead in terms of controllable and uncontrollable errors: systematic errors are potentially under your control (given a particular experimental setup), whereas random errors aren't. Should you make a distinction then between controllable (cycling cuvette in and out of the holder) and potentially controllable errors (dilution)? And the latter may then become controllable with a different experimental setup? Cheers, Ed. -- I don't know why the sacrifice thing didn't work. Science behind it seemed so solid. Julian, King of Lemurs
Re: [ccp4bb] statistical or systematic? bias or noise?
Pete, On Mon, 2013-03-11 at 13:42 -0500, Pete Meyer wrote: My take on it is slightly different - the difference seems to be more on how the source of error is modeled (although that may dictate changes to the experiment) rather than essentially depending on how the experiment was conducted. Or (possibly) more clearly, systematic error is a result of the model of the experiment incorrectly reflecting the actual experiment; measurement error is due to living in a non-deterministic universe. I see your point. I want to clarify that reproducing an experiment as far back as possible is best. Of course it's possible to design an experiment better and account for pipetting errors. The question is not whether it has to be done (certainly yes) but whether pipetting error should be considered as inaccuracy or imprecision when the experiment is not repeated. One can say it's inaccuracy when it is not estimated and imprecision when it is. Or one can accept Ian's suggestion and notice that there is no fundamental difference between things you can control and things you can potentially control. IIUC, you are saying that nature of the error should be independent of my decision to model it or not. Other words, if I can potentially sample some additional random variable in my experiment, it contributes to precision whether I do it or not. When it's not sampled, the precision is simply underestimated. Does that make more sense? Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] [Err] Re: [ccp4bb] statistical or systematic? bias or noise?
By the way, am I the only one who gets this thing with every post? If anyone can ask Jin Kwang (liebe...@korea.ac.kr) to either clean up his mailbox or unsubscribe, that would be truly appreciated. Delete button is easy and fun to use, but this has been going on for quite some time. On Tue, 2013-03-12 at 04:16 +0900, spam_mas...@korea.ac.kr wrote: ransmit Report: liebe...@korea.ac.kr ; 5 õ Ͽ4ϴ. ( / : 554 Transaction failed. 402 Local User Inbox Full (liebe...@korea.ac.kr) 4,61440,370609(163.152.6.98)) / User unknown :; ڰ x = Socket connect fail: DATA write fail: ۽ DATA reponse fail : κ -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
On 03/04/2013 10:02 AM, Marcin Wojdyr wrote: It also puzzled me, but I haven't done more careful benchmarking yet. What did you get after compiling refmac? My numbers are not as impressive, but I also get quite detectable improvement, from 35s to 27s (ccp4-6.3.0 vs compiled from source). This is with gcc-4.6 though. Importantly, both binaries are the *same* version of refmac, 5.7.0032. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
Thierry, I ran both versions on the same input file and numerical results are essentially the same. After 10 cycles of refinement the r.m.s.d. between two models produced with different versions is 0.0004A. Basically, about 5% of coordinates differ in the last digit (i.e. by 0.001A). Frankly, I expected results to be exactly identical, but the difference is too small to be of concern, imho. Cheers, Ed. On 03/04/2013 10:19 AM, Fischmann, Thierry wrote: Ed, Are the numerical results the same ? Not likely that there is a problem. But if you haven't done it already it is worth checking by running the tests provided with the suite. Aggressive optimization can be a source of bugs. Best regards Thierry -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Monday, March 04, 2013 8:55 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04 On Mon, 2013-03-04 at 11:37 +, Marcin Wojdyr wrote: Running times were, correspondingly, 32.2s, 35.1s and 18.7s. Numbers are almost too impressive to believe :) How does it compare with ifort (which I thought should be the fastest option on intel processors and thus unavailable (not free) for most DIY compilation given licensing issues)? -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
Adam, On Mon, 2013-03-04 at 09:56 +, Adam Ralph wrote: One of the first routines called by CCP4 progs is ccp4fyp. This initialises the CCP4 environment. I think you might have missed in my original post that I get an error when I *do* source ccp4 environment. Does the error occur with refmac alone or with every CCP4 prog? I cannot tell because I am compiling only refmac, not all of the ccp4. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
Indeed, the problem goes away when -static flag is omitted. Interestingly, the resulting binary dependencies do not include any ccp4-related libraries. For those interested, I was able to track the segfault down to the close() operator - so basically it fails when closing a file opened with ccpdpn routine. At that point I had a lucky guess of removing the flag (somewhat inspired by noticing that refmac5 binary from cc4-6.3.0 is dynamic and after trying to compile separately a short piece of code that only opened and closed a file), so the issue is solved as far as my goals are concerned. On Mon, 2013-03-04 at 10:04 +, Garib N Murshudov wrote: Dear all I think this error has been dealt with (Ed will correct me if I am wrong). The problem was -static in compilation. For whatever reason in some gcc (gfortran) -static does not work (it compiles but has problems in running, what is the reason is not clear to me). Sometimes in later gcc -static-libgcc -static-libgfortran works but not always. These flags are needed for distribution purposes. If you are compiling and using on the same computer then you should not need it. regards Garib On 4 Mar 2013, at 09:56, Adam Ralph wrote: Dear Ed, The error does indeed happen in ccp4lib. One of the first routines called by CCP4 progs is ccp4fyp. This initialises the CCP4 environment. See lib/cctbx/cctbx_sources/ccp4io/lib/src/ccp4_general.c. If you look at the code you can see that $CINCL is determined at run-time. You are right that this environment var is not needed at compile time. Files like environ.def and default.def are read at this time. Perhaps there has been a corruption of one of these files or you are pointing to an earlier version of $CINCL. Does the error occur with refmac alone or with every CCP4 prog? Adam Dr Garib N Murshudov Group Leader, MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
On Mon, 2013-03-04 at 11:37 +, Marcin Wojdyr wrote: Running times were, correspondingly, 32.2s, 35.1s and 18.7s. Numbers are almost too impressive to believe :) How does it compare with ifort (which I thought should be the fastest option on intel processors and thus unavailable (not free) for most DIY compilation given licensing issues)? -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] compiling refmac5 on Ubuntu 12.04
Adam, I am not compiling CCP4, just refmac. IIUC, all that sourcing ccp4.setup does is it sets $CLIB for refmac makefile to find libccp4c and libccp4f. And presumably lapack and libblas, but that's a separate issue. On Thu, 2013-02-28 at 10:28 +, Adam Ralph wrote: Hi Ed, It looks as though you have not sourced $CCP4/include/ccp4.setup. This needs to be customized and sourced before you configure and make CCP4. Adam -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
[ccp4bb] compiling refmac5 on Ubuntu 12.04
I am trying to compile refmac from source on a machine running Ubuntu 12.04. In a nutshell, after some troubleshooting I end up with executable that generates a segmentation fault. Log-file states that CCP4 library signal ccp4_parser:Failed to open external command file (Success) raised in ccp4_parser (hardly a success). Potentially relevant details are that I had to compile libccp4 and libmmdb to get to this point. If I don't configure the CCP4, I get this when trying to run refmac CCP4 library signal ccp4_general:Cannot open environ.def (Error) raised in ccp4fyp refmacgfortran: Cannot open environ.def refmacgfortran: Cannot open environ.def So perhaps it's some incompatibility between libccp4/libmmdb that I compiled and those that came with CCP4 installation (by the way, the new update feature rocks indeed). But I tried lifting these libraries from CCP4 installation when compiling refmac and I get the same segmentation fault. Any suggestions for troubleshooting/advice on how to compile refmac from source are appreciated. Refmac version is 5.7.0032. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Link problem with Refmac.
We might have just found a new recurring discussion - what to do with insertion codes! I am sure the opinion split is close to 50/50. Personally, I don't think insertion codes make sense in the first place. Are catalytic triad residues always the same distance from the N-terminus? No. The residue number designates its position in the sequence, not its relationship to other like-minded enzymes. That is a useful (as it defines peptide bonds) and consistent definition. Even with antibodies, where these can be indeed interpreted as insertions, the mess is enormous since only half the structures conform to WuKabat numbering (and there are two of those). It is true however that it's just as easy to implement sorting that pays attention to insertion codes as it is structural alignment based residue matching. Cheers, Ed. Original message From: herman.schreu...@sanofi.com Date: To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Link problem with Refmac. I fully second this. The treatment of insertion codes of many programs is a mess, leaving you with the option to renumber (and loose contact with often a huge body of existing literature), or stuff the pdb with link and gap records (if recognized at all by the program used). It would be a great help if the programmers would use a simple distance criterion (e.g. N - C distance 2.0 A) to decide whether amino acids are linked instead of forcing a link between residues which are more than 10 A apart as in the current case. Cheers, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Monday, February 18, 2013 10:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Link problem with Refmac. Hi Ian, I avoid renumbering whenever I can. If I do have to renumber things (e.g. to get proper connectivity in PDB entry 2j8g), I do it by hand. So no help there. As for dealing with insertion codes in general, why not try to convince the developers of the 'brain-damaged' to support insertion codes? I've asked quite a few for these sort of updates and many were very helpful. The problem is that most developers discover the existence of insertion codes after they set up a data structure for the coordinates. Adding support afterwards can be quite a hassle. The more users ask for such support, the more likely it will be implemented. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle Sent: Monday, February 18, 2013 19:40 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Link problem with Refmac. Hi Robbie OK I just realised what's going on. In my script I renumber the input PDB file (starting at 1 for each chain and incrementing by 1) and keep the mapping so I can renumber it back afterwards for human consumption. So you're completely correct: there is indeed a residue A59 after renumbering! This is to avoid headaches with brain-damaged programs that can't cope with insertion codes and residue numbers out of sequence. So I guess I'm going to have to be smarter in my renumbering program and make sure I maintain any increasing gaps in the numbering which indicate real gaps in the sequence and only renumber over insertions and decreasing gaps. It doesn't actually matter what the new numbers are since the user never sees them. But this must be a common problem: how do others handle this? E.g. pdbset blindly renumbers with a increment of 1 (and anyway it doesn't renumber any LINK, SSBOND CISPEP records as I do) so it would have the same problem. Cheers -- Ian On 18 February 2013 17:09, Robbie Joosten robbie_joos...@hotmail.com wrote: Hi Ian, The warning refers to a MET 59 in chain A whereas you only have MET 72. That is very suspicious. Non-sequential residues further apart than x Angstrom automatically get a gap record. Have you tried a newer version of Refmac, because this feature was added quite a while ago? What is your setting for 'MAKE CONN' when you run Refmac? Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ian Tickle Sent: Monday, February 18, 2013 17:32 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Link problem with Refmac. All, I'm having a problem with Refmac (v. 5.7.0025) that I don't understand. It's linking 2 residues that it shouldn't be. Here's the relevant message in the log file: WARNING : large distance for conn:TRANS dist = 10.768 ch:AA res: 58 THR -- 59 MET ideal_dist= 1.329 Note that there are no LINK (or LINKR) records in the PDB header. Here are the input co-ords for the relevant residues (not linked): ATOM 887 N THR A 58 13.587 1.365 19.814 1.00 14.28 A N ATOM 888 CA THR A 58 14.743 1.126
Re: [ccp4bb] Renumbering and retaining state information pdb file
There should be many ways to do this. You can split the file, renumber with pdbset, and then reassemble it. This may be useful http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Useful_scripts_(aka_smart_piece_of_code) Cheers, Ed Original message From: Amar Joshi aj...@leicester.ac.uk Date: To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Renumbering and retaining state information pdb file Hi, I am trying to tidy up an old NMR structure (not mine) and I want to renumber the residues in all the states. When I use pdbset starting at X, the numbers are changed but the state information is lost. How can I retain the state in my modified pdb file? Thanks, Amar - Amar Joshi Ph.D Bayliss Lab Department of Biochemistry Henry Wellcome Building University of Leicester Lancaster Road, Leicester LE1 9HN Tel: 0116 229 7082 Email: aj...@leicester.ac.uk --
Re: [ccp4bb] S-nitrosylation protein
Maybe you can try different energies hoping that damage is wavelength dependent. It must be dose dependent though, so you may consider merging short sweeps from multiple crystals. Original message From: Uma Ratu rosiso2...@gmail.com Date: To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] S-nitrosylation protein Dear All: I plan to use X-ray crystallography method to study the S-nitrosylated protein structure. The native protein crystals diffracted to 2A with synchrontron. I now have the crystals of S-ntrosylated protein. Since S-NO moiety appears to be unstable to synchrotron radiation, could you advice / comments on the stratage on the data collection of S-nitrosylated protein crystals? The protein crystals did not diffract well with in house X-ray. Thank you for your comments. Uma
Re: [ccp4bb] refmac5 MMA bug
On Mon, 2013-02-11 at 09:56 +0100, Robbie Joosten wrote: This is a 'compatability' option in Refmac that internally renames atoms. If you comment out 'MMA .C7 CM' in your mon_lib_list.cif file, the problem will disappear. Robbie, thanks a lot - this fixes it. Is this still considered a bug? From what I understand, the data_comp_synonym_atom_list entry indicates that whenever MMA C7 atom is encountered, it will be internally renamed to CM. However, the $CCP4_LIB/data/monomers/m/MMA.cif should then refer to CM as well. But that cif-file still uses C7. Maybe this gets fixed in ccp4 updates, which reminds me to get that set up at last. Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
[ccp4bb] refmac5 MMA bug
I see a strange issue with a model that includes O1-methyl-mannose (three letter code MMA). Basically, refmac fails and says that C7 is missing in the model while CM is absent from the library. The problem is that there is no CM atom in the pdb file, while C7 is right there. This happens with Refmac_5.7.0029, and I see no obvious issues with the corresponding cif-file in the monomer library. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
Patrick, Something related: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Conditions_prone_to_salt_crystallization Truth be told, we recently had a major breakthrough with the peg/fluoride condition I came to consider a useless salt crystal generator. So tables like these are undoubtedly useful but do not reduce workload. :) This is also a rather interesting finding http://www.google.com/url?sa=tsource=webcd=12ved=0CDEQFjABOAourl=http%3A%2F%2Fwww.aseanbiotechnology.info%2FAbstract%2F21021153.pdfei=N_kUUYfkBafV0gG09ICoAgusg=AFQjCNE7C-m6IkLPUay9gEEM60yJF46ZQg Basically, presence of protein may induce salt crustallization. To me, this means that diffraction pattern is the best indicator. Frank already said exactly that, of course. Cheers, Ed. Original message From: Patrick Shaw Stewart patr...@douglas.co.uk Date: To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals Good morning Frank On a related idea, do you typically use a limited number of buffers (buffer plus salt) for the final purification step of your proteins? If so, do you have a chart of where salt crystals may appear in the screens that you use most often? Could you put that chart on your web site to help the community? People could pick one of your standard buffer mixes to make their lives easier later on. Best wishes Patrick On 8 February 2013 07:18, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Test the diffraction - that's the only way. But given the other junk in the drop, chances are they're salt. (And don't post 5Mb attachments, please.) On 07/02/2013 22:24, amro selem wrote: Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 09:13 -0500, Edward A. Berry wrote: I like to take a 5-sec 180* oscillation which gives plenty of spots in a nice pattern for a salt crystal Second that It also confuses bystanders really well - what a strange diffraction pattern - half salt (small unit cell) / half protein (lots of spots). Takes your mind off the gloom of your dashed hopes :) -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 14:53 +0400, Evgeny Osipov wrote: Protein crystals behave rather as gelatine and not as solid I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] protein crystals or salt crystals
On Fri, 2013-02-08 at 09:57 -0500, Jacob Keller wrote: do you have a reference quickly on hand http://www.ncbi.nlm.nih.gov/pubmed/8129868 and references therein http://www.sciencedirect.com/science/article/pii/S0022024801010922 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1300955/ The last reference is not strictly speaking about protein crystals, but also emphasizes how protein as elastic material is more of a solid (with some peculiar properties likely arising from large entropic contribution to its deformation energy. -- Bullseye! Excellent shot, Maurice. Julian, King of Lemurs.
Re: [ccp4bb] protein crystals or salt crystals
Michael, It seems to me we have no disagreement, as we both say that it is *unusual* for protein crystals to be non-fragile. Furthermore, my objection is to gelatin characterization. I may be, as is my custom, wrong, but in terms of elasticity gels are purely entropic. Protein crystals, even the malleable ones you describe, have both enthalpic and entropic components to the deformation free energy (admittedly the entropic component is stronger than in regular materials). Your comment on the PEG-induced slow cross-linking is very interesting. What you describe fits perfectly the behavior of long rods of a deliberately cross-linked crystal - they bend and don't snap. Protein crystals (at least some) are also characterized by an unusually broad range of linear response to mechanical deformation, up to 10% http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143058/ According to the theory in this paper though, you might be destroying diffraction by bending the crystal, since in crystal denaturation is likely irreversible. From what you are describing (60 degrees, 150x40x40 um), the linear deformation should reach almost 50%, definitely enough for partial crystal denaturation. Cheers, Ed. On Fri, 2013-02-08 at 11:22 -0500, R. M. Garavito wrote: Ed, Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. I hate to disagree with the disagreement, but there are many exceptions to this rule. I have seen many protein crystals that are quite malleable and bendable. One protein produced rod-shaped crystals (150x40x40 um) that I could bend by almost 60 degrees and it would slowly snap back. Mounting it old school was a real pain, and their diffraction was mediocre. However, the majority of the crystals I have worked with adhere to the general rule you describe. Where the crystal physical behavior is anomalous, it is often when PEG is used and/or there are multiple components that contribute to the crystal's integrity (as in the case of membrane protein crystals with detergent). In the former case, crystals that sit in PEG solutions too long tend to be cross-linked (most likely due to the aldehydes that can exist in some batches of PEG). One could argue that the crosslinking adds long-range elasticity and a resistance to fracturing. In the latter case, I have observed large beautiful crystals of membrane proteins that have the consistency and malleability of warm butter. Sometimes optimization improved their integrity, and other times a new crystal form is needed. Regards, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Feb 8, 2013, at 9:23 AM, Ed Pozharski wrote: On Fri, 2013-02-08 at 14:53 +0400, Evgeny Osipov wrote: Protein crystals behave rather as gelatine and not as solid I'd have to disagree on that. Protein crystals are fragile but not soft. If your crystals are like gelatine it's unusual. It has been demonstrated that elastic properties of protein crystals are similar to organic solids. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] gedit on mac terminal
Try this http://www.mac-forums.com/forums/os-x-apps-games/239657-gedit-command-not-found-terminal.html Original message From: LISA science...@gmail.com Date: To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] gedit on mac terminal Hi all, I installed gedit on my mac applications. But i can not use it by terminal. Please help me figure it out. Thanks. lisa
[ccp4bb] engh huber
To what extent modern geometric restraints have been upgraded over original EnghHuber? And where I can find a consensus set of values (with variances)? For example, Fisher et al., Acta D68:800 discusses how histidine angles change with protonation, and refers to EnghHuber when it says that ND1-CE1-NE2 goes from 111.2 to 107.5 when histidine acquires positive charge (Fig.6). But angle table (Table 3) in original EnghHuber from 1991 does not have any 107.5 value and seems to suggest that the numbers should rather be 111.7+-1.3 and 108.4+-1.0, respectively. I understand that these values are derived from structural databases and thus can be frequently updated. Is there some resource where most current values would be listed? Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] engh huber
Article in the Tables is the answer to my question about the latest EnghHuber parameters. These still don't match Fig.6 from Fisher, but I am OK with using Tables for my internal purposes. Thanks to Mitchell and Dale for prompt response. Cheers, Ed. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] How to merge data from 2 separate sections of same crystal
On 12/10/2012 08:45 PM, Yuri Pompeu wrote: hello everyone, I have collected data on a problematic crystal. (first mistake...) Images spanning phi angles 45-80 look ok and usable, also images 229-279 are usable (index well and merge well too). How can I combine the 2 separate .mtz files from Mosflm when I scale them? Attempts to process them in the same session keep maling the program crash! Thank you very much Did you try the 85 frames to mosflm in one session (maybe move bad files away from the current folder for a clean try). -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] thanks god for pdbset
Francois, I did not realize Phil Evans is god (perhaps a minor one as he did not yet earn a capital G). I do concur that insertion code is evil. I had to re-refine an old antibody structure recently and it messes up coot sequence window and breaks refmac bond restraints. Evil, evil,.evil. Cheers, Ed. On Wed, 2012-12-05 at 16:58 +0900, Francois Berenger wrote: Especially the renumber command that changes residue insertion codes into an increment of the impacted residue numbers. Regards, F. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] thanks god for pdbset
On Wed, 2012-12-05 at 17:02 +0100, Robbie Joosten wrote: Does 128A come before or after 128? Robbie, shouldn't it simply depend on which residue record comes first in the pdb file? Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Mg++ interactions
On Tue, 2012-11-27 at 21:46 -0800, William G. Scott wrote: Are Mg++ ions ever observed to chelate primary amines? MESPEUS reports, for example, 13 structure where magnesium is coordinated by a lysine. 7 with arginines and a bunch of asn/gln side chains as well. It does not prove, of course, that primary amines coordinate magnesium ions, only that in a limited number of cases crystallographers have interpreted their data this way. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
