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Re: [phenixbb] DTT mediated disulfide crosslink across a crystallographic two-fold! (Pavel Afonine)
by Joseph Noel 04 Aug '10

04 Aug '10

Thanks Pavel! I will wait and see what Ralf says regarding defining the symmetry operator. If I am using the GUI for phenix.refine should I create a new file with the bond definition you mention and include it as one of the starting files or should I edit the Refine_*.eff file that gets created and insert it somewhere there? ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________ On Aug 4, 2010, at 10:22 AM, phenixbb-request(a)phenix-online.org wrote: > Send phenixbb mailing list submissions to > phenixbb(a)phenix-online.org > > To subscribe or unsubscribe via the World Wide Web, visit > http://phenix-online.org/mailman/listinfo/phenixbb > or, via email, send a message with subject or body 'help' to > phenixbb-request(a)phenix-online.org > > You can reach the person managing the list at > phenixbb-owner(a)phenix-online.org > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of phenixbb digest..." > > > Today's Topics: > > 1. Re: DTT mediated disulfide crosslink across a > crystallographic two-fold! (Joseph Noel) > 2. Re: high average b-factor vs. Wilson B (Gino Cingolani) > 3. Re: high average b-factor vs. Wilson B (Frank von Delft) > 4. Re: DTT mediated disulfide crosslink across a > crystallographic two-fold! (Pavel Afonine) > 5. Re: high average b-factor vs. Wilson B (Pavel Afonine) > 6. r-work and r-free for specific resolution range(s) > (Kay Diederichs) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 4 Aug 2010 04:53:25 -0700 > From: Joseph Noel <noel(a)salk.edu> > To: phenixbb(a)phenix-online.org > Subject: Re: [phenixbb] DTT mediated disulfide crosslink across a > crystallographic two-fold! > Message-ID: <91F9FC5E-00E4-454C-AB99-2DD353BDC4A5(a)salk.edu> > Content-Type: text/plain; charset="us-ascii" > > Hi Ralf and Pavel, > > Unfortunately, Phenix didn't recognize a disulfide bond should exist between the sulfur atoms of DTT and the side chain sulfur atom of the cysteine residue in question. I position everything well in the Fo-Fc and 2Fo-Fc maps and ensured the two sulfurs were approximately 2.1 A apart. However, after refinement the DTT molecule moved so the two sulfurs in question where nearly 3 A away. Maybe this is because it is a mixed disulfide between a DTT molecule and a side-chain Cys rather then what most programs expect which is a disulfide between two Cys residues. Is it possible I need to define the complete chemical entity as a hetatm (the disulfide linked Cys residue? Is there a way to specify that a covalent bond should exist between the SG atom of the Cys in question and the S atoms of DTT with DTT lying across the crystallographic two-fold given its internal 2-fold symmetry? > > Joe > ___________________________________________________________ > Joseph P. Noel, Ph.D. > Investigator, Howard Hughes Medical Institute > Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics > The Salk Institute for Biological Studies > 10010 North Torrey Pines Road > La Jolla, CA 92037 USA > > Phone: (858) 453-4100 extension 1442 > Cell: (858) 349-4700 > Fax: (858) 597-0855 > E-mail: noel(a)salk.edu > > Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 > Web Site (HHMI): http://hhmi.org/research/investigators/noel.html > ___________________________________________________________ > >

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Re: [phenixbb] DTT mediated disulfide crosslink across a crystallographic two-fold!
by Joseph Noel 04 Aug '10

04 Aug '10

Hi Ralf and Pavel, Unfortunately, Phenix didn't recognize a disulfide bond should exist between the sulfur atoms of DTT and the side chain sulfur atom of the cysteine residue in question. I position everything well in the Fo-Fc and 2Fo-Fc maps and ensured the two sulfurs were approximately 2.1 A apart. However, after refinement the DTT molecule moved so the two sulfurs in question where nearly 3 A away. Maybe this is because it is a mixed disulfide between a DTT molecule and a side-chain Cys rather then what most programs expect which is a disulfide between two Cys residues. Is it possible I need to define the complete chemical entity as a hetatm (the disulfide linked Cys residue? Is there a way to specify that a covalent bond should exist between the SG atom of the Cys in question and the S atoms of DTT with DTT lying across the crystallographic two-fold given its internal 2-fold symmetry? Joe ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________

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DTT mediated disulfide crosslink across a crystallographic two-fold!
by Joseph Noel 04 Aug '10

04 Aug '10

Hi Phenix Users, I have found a beautifully ordered DTT molecule stretching across a crystallographic two-fold and linking two symmetry related cysteine side chains. Since part of this molecule sits on across the two-fold (special position) I will specify the occupancy as 0.5. I am hoping to get some information on how easy (or how difficult) it is to specify this arrangement in Phenix for refinement since it is covalent and sitting across a crystallographic two-fold (and presumably stabilizing the lattice). Will ReadySet build the appropriate CIF file if I build the DTT into the density and give it a 0.5 occupancy? Joe ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________

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Re: [phenixbb] Adequate size for Free R test set?
by Joseph Noel 03 Aug '10

03 Aug '10

Pavel, Thanks so much for the suggestions. They are really helpful! A few questions relative to Phenix. Is there a way to check with Phenix the thin resolution shells? I created Free Rs in Phenix using the thin resolution shells options but removed the 2000 limit and instead used 5%. Maybe I over-did it relative to your reply. Just wondering how to check each of these thin shells. Alternatively, does Phenix ensure one has an adequate number of test reflections in each "thin shell" if this option is set? If so, what should I used for the maximum number of reflections and/or %? Thanks again! Joe ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________ On Aug 3, 2010, at 11:02 AM, phenixbb-request(a)phenix-online.org wrote: > Send phenixbb mailing list submissions to > phenixbb(a)phenix-online.org > > To subscribe or unsubscribe via the World Wide Web, visit > http://phenix-online.org/mailman/listinfo/phenixbb > or, via email, send a message with subject or body 'help' to > phenixbb-request(a)phenix-online.org > > You can reach the person managing the list at > phenixbb-owner(a)phenix-online.org > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of phenixbb digest..." > > > Today's Topics: > > 1. Re: Adequate size for Free R test set? (Pavel Afonine) > 2. Re: follow up on message 2: phenix.maps --Message: not > implemented (amadrona(a)uci.edu) > 3. Re: follow up on message 2: phenix.maps --Message: not > implemented (Pavel Afonine) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 03 Aug 2010 10:38:57 -0700 > From: Pavel Afonine <pafonine(a)lbl.gov> > To: phenixbb(a)phenix-online.org > Cc: Joseph Noel <noel(a)salk.edu> > Subject: Re: [phenixbb] Adequate size for Free R test set? > Message-ID: <4C585431.7000107(a)lbl.gov> > Content-Type: text/plain; charset="iso-8859-1"; Format="flowed" > > Hi Joe, > > I think almost every one has his/her own opinion on this... Here is what > I think: > > 1) The test set should be such that each "relatively thin resolution > shell" receives at least 50 reflections, and we empirically found that > 150 is "good enough" withing phenix.refine framework. > For "relatively thin resolution shell" definition see: > Lunin & Skovoroda. Acta Cryst. (1995). A51, 880-887. "R-free > likelihood-based estimates of errors for phases calculated from atomic > models". > > This basically defines how many test reflections you need. > > 2) It is customary to set aside either 5 or 10% for test set, with the > total maximum 2000. These are all "magic numbers", that I presume more > or less satisfy "1)" so they became widely used. > > 3) Presence of high-order NCS and selecting free-flags using "thin > shells" algorithm is a different story (Acta Cryst. (2006). D62, > 227--238). It is good to do that because it removes the cross-talk > between test and work reflections due to NCS, but at the same time it > invalidates the requirement "1)". So, this is a gray area (for me at least). > > 4) Some people believe that the final refinement run should be done > using all reflections, arguing that taking away 5-10% of test > reflections worsens the maps. There is some truth in this, yes, removing > the data worsens the maps, but: > a) it is noticeable (in a sense that it can reduce the interpretability > of some parts of the map) only in extreme cases of somewhat low > resolution or low completeness data, b) in most of all other cases it is > simply negligible, c) removing reflections randomly has much smaller > effect than removing them systematically (see page #40 here: > http://www.phenix-online.org/presentations/latest/pavel_maps.pdf and > some relevant references in 2010 PHENIX paper in Acta D). However, if > you do that "final run", you will invalidate the final refinement > statistics, Rfree and Rwork, and thus obtained final structure cannot > have the Rfree associated with it anymore. > > Pavel. > > > On 8/3/10 10:04 AM, Joseph Noel wrote: >> Hi Folks, >> >> Its been a while since I personally refined many structures. In the >> past, I used as a default, 5% of my unique reflections for the Free R >> test set. I have a high resolution structure with 150,000 unique >> reflections and noticed that Phenix defaults are 5% or 2000 >> reflections which ever is smaller. What is the current consensus on an >> adequate number of unique reflections to use for cross-validation? >> >> Thanks! >> Joe >> >> P.S. I really, really love Phenix. >> ___________________________________________________________ >> Joseph P. Noel, Ph.D. >> Investigator, Howard Hughes Medical Institute >> Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics >> The Salk Institute for Biological Studies >> 10010 North Torrey Pines Road >> La Jolla, CA 92037 USA >> >> Phone: (858) 453-4100 extension 1442 >> Cell: (858) 349-4700 >> Fax: (858) 597-0855 >> E-mail: noel(a)salk.edu <mailto:[email protected]> >> >> Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 >> Web Site (HHMI): http://hhmi.org/research/investigators/noel.html >> ___________________________________________________________ >> >> >> _______________________________________________ >> phenixbb mailing list >> phenixbb(a)phenix-online.org >> http://phenix-online.org/mailman/listinfo/phenixbb > >

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Adequate size for Free R test set?
by Joseph Noel 03 Aug '10

03 Aug '10

Hi Folks, Its been a while since I personally refined many structures. In the past, I used as a default, 5% of my unique reflections for the Free R test set. I have a high resolution structure with 150,000 unique reflections and noticed that Phenix defaults are 5% or 2000 reflections which ever is smaller. What is the current consensus on an adequate number of unique reflections to use for cross-validation? Thanks! Joe P.S. I really, really love Phenix. ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________

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New version complaint
by Simon Kolstoe 03 Aug '10

03 Aug '10

Hi there, I've been using version 1.6.2-432 on a Macbook and was particularly enjoying the "Show Histograms" button on the POLYGON results page. However today I updated to version 1.6.4-486 using the dmg installer, and that useful option has disappeared! Is there a good reason for the absence of this function, and if not can we get it back? Thanks, Simon --------------------------------------------------------------- Dr Simon Kolstoe Laboratory for Protein Crystallography Centre for Amyloidosis & Acute Phase Proteins UCL Medical School Rowland Hill Street, London NW3 2PF Tel: 020 7433 2765 http://www.ucl.ac.uk/~rmhasek --------------------------------------------------------------

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Re: [phenixbb] Phenix Autobuild
by Thomas C. Terwilliger 03 Aug '10

03 Aug '10

Hi Munan Shaik, Phenix autobuild can read several types of sequence files, but the basic format is very simple: enter one copy of the sequence of each chain, separated by a blank line or a line starting with ">" between chains. You can optionally add any lines starting with ">" which will be ignored. So here is an example: >>> alpha subunit (type 1) MTMPYYASAEQIMRDRSELARKGIARGRSVVVLTFRDGVLFVAENPSTALHKVSELYDRL GFAAVGKYNEFENLRRAGIVHADMRGYSYDRRDVTGRSLANAYAQTLGTIFTEQPKPYEV EICVAEVGRVGSPKAPQLYRITYDGSIVDEQHFVVMGGTTEPIATAMRESYRADLDLEAA VGIAVNALRQGGAGEGEKRNVDVASLEVAVLDQSRPRRAFRRIAGTALEQLVPAEPAAAS ESAPEPKPDTETKPADTQD >>> beta subunit (type 1) MTADRPALRTGDRDTRLSFGSNLSSFTDYLRGHAPELLPENRIGHRSHSTRGGDGMESGD LAPHGTTIVALTYKGGVLLAGDRRATQGNLIASRDVEKVYVTDEYSAAGIAGTAGIAIEL VRLFAVELEHYEKIEGVPLTFDGKANRLASMVRGNLGAAMQGLAVVPLLVGYDLDADDES RAGRIVSYDVVGGRYEERAGYHAVGSGSLFAKSALKKIYSPDSDEETALRAAIESLYDAA DDDSATGGPDLTRGIYPTAVTITQAGAVHVSEETTSELARRIVAERTEQGGSAR Please let me know if that doesn't do it! All the best, Tom T >> Hi everybody, >> I have a problem with Phenix autobuild. I want to input sequence file >> together >> with the pdb and mtz that I got from molecular replacement. But I have no >> idea >> which types of sequence files its required. if anybody please help me out. >> >> >> thanks >> =================================================== >> Md. Munan Shaik >> Department of Chemical Biology >> University of Padova >> via G. Colombo 03 >> Padova 35131, Italy >> >> >>

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coot links on Mac
by Joseph Noel 02 Aug '10

02 Aug '10

Dear Phenix Team, I noticed that coot behaves unusually on the MacOS version of phenix. It will open when you hit the button in the GUI but it will not open any of the files you specify. Just the main coot window which you then have to load everything as you would normally if running coot stand alone. Also, I noticed that when you run coot from the gui link on MacOS that after manipulating the structure for rebuilding, etc it will freeze requiring a force quit of x-windows to recover. However, if I open coot directly from my xterm on MacOS it works fine and no freezes. Just a note. Love the program! Joe ___________________________________________________________ Joseph P. Noel, Ph.D. Investigator, Howard Hughes Medical Institute Professor, The Jack H. Skirball Center for Chemical Biology and Proteomics The Salk Institute for Biological Studies 10010 North Torrey Pines Road La Jolla, CA 92037 USA Phone: (858) 453-4100 extension 1442 Cell: (858) 349-4700 Fax: (858) 597-0855 E-mail: noel(a)salk.edu Web Site (Salk): http://www.salk.edu/faculty/faculty_details.php?id=37 Web Site (HHMI): http://hhmi.org/research/investigators/noel.html ___________________________________________________________

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Phenix version 1.6.4 released
by Paul Adams 31 Jul '10

31 Jul '10

The Phenix developers are pleased to announce that version 1.6.4 of Phenix is now available. Binary installers for Linux, and Mac OSX platforms are available at the download site: http://phenix-online.org/download/ Just some of the new features in this version (1.6.4) are: - GUI: - new interfaces for eLBOW, CIF to MTZ conversion, phenix.find_ncs, phenix.superpose_maps - new structure comparison tool (evaluation of multiple related structures) - supports execution of jobs on the Sun Grid Engine queueing system - maps for PyMOL now output in (smaller, faster) CCP4 format - display of sequence and secondary structure in wizards and validation - new wide-format windows for wizards and phenix.refine - many bug fixes - Automated model building: - phenix.autosol now much faster and improved model-building after density modification - phenix.phase_and_build (carries out an iterative process of building a model as rapidly as possible and uses this model in density modification to improve the map. Up to 10x faster compared to AutoBuild, but model quality is nearly as good) - phenix.build_one_model (quickly build a single model from a map and sequence file, or extend an existing model) - phenix.assign_sequence (carry out an improved sequence assignment of a model that you have already built. Uses the new loop libraries available in phenix.fit_loops) - phenix.apply_ncs (apply NCS operators to a single copy of a protein to create all the NCS‐related copies) - phenix.find_ncs (now supports identification of NCS operators from a density map) - phenix.fit_loops (two main algorithms now available - fit short gaps using a loop library derived from high‐resolution structures in the PDB, or build loops directly by iterative extension with tripeptides - phenix.refine: - reference model restraints (restraint dihedral angles to a higher resolution reference model) - alternate ideal angles for torsion definitions (preserves side chain rotameric states) - automatic flipping of incorrect N/Q/H rotamers - custom planarity restraints - hydrogen-bond restraints for Watson-Crick base pairs - reflections with Fobs=0 used in refinement - automatic optimization of mask parameters - map parameters now consistent with phenix.maps - new custom_nonbonded_symmetry_exclusions atom selection parameter to fine tune non-bonded interactions across symmetry axes - Phasing: - Option for Phaser likelihood scoring of substructure solutions in HySS - SOLVE converted to C++; FORTRAN compiler no longer required for Phenix compilation - Other: - KiNG kinemage viewer incorporated into Phenix - Real space refinement option in ligand fitting - B-factor sharpening option for maps For a full list of changes see: http://www.phenix-online.org/documentation/CHANGES Please note that there is a new publication that should be used to cite use of Phenix: PHENIX: a comprehensive Python-based system for macromolecular structure solution. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger and P. H. Zwart. Acta Cryst. D66, 213-221 (2010). Full documentation is available here: http://www.phenix-online.org/documentation/ There is a Phenix bulletin board: http://www.phenix-online.org/mailman/listinfo/phenixbb/ Please consult the installer README file or online documentation for installation instructions. Direct questions and problem reports to the bulletin board or: help(a)phenix-online.org and bugs(a)phenix-online.org Commercial users interested in obtaining access to Phenix should visit the Phenix website for information about the Phenix Industrial Consortium. The development of Phenix is principally funded by the National Institute of General Medical Sciences (NIH) under grant P01-GM063210. We also acknowledge the generous support of the members of the Phenix Industrial Consortium. -- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology Building 64, Room 248 Tel: 1-510-486-4225, Fax: 1-510-486-5909 http://cci.lbl.gov/paul Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. Executive Assistant: Marian Harris [ MSHarris(a)lbl.gov ] [ 1-510-486-6886 ] --

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