- phenixbb - phenix-online.org

structure factor labels in phenix.refine
by Tanner, John J. 08 Nov '13

08 Nov '13

I have two questions about which structure factor amplitudes are refined against in phenix.refine. 1. Which structure factor amplitude is used in refinement when the labels in the .def file are the following? labels = "F-obs(+),SIGF-obs(+),F-obs(-),SIGF-obs(-)" 2. Is anomalous data used in the refinement when the labels are as follows? labels = "F,SIGF,DANO,SIGDANO,ISYM" I ask these questions because I am wondering which structure factor amplitudes to deposit, and I am trying to rationalize differences between R-factors from refinement and Generate Table 1. Thanks. Jack Tanner John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: tannerjj(a)missouri.edu<mailto:[email protected]> http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html

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few questions about resolving new structure through MR
by Zhihong Yu 07 Nov '13

07 Nov '13

Hi, all I'm a rookie in resolving a brand new structure. I have some questions for my current case and look forward to some suggestions. Now I’m working on a protein like this: N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a diffraction data just to 3.5Å, and there is no complete homology structure in pdb bank, but only a homology structure (named as structureX later) for domainB with ~30% sequence identity, so I have some questions as following: 1. Is it possible to find a resolution through MR approach using structureX as a search model? Especially considering that the resolution is only 3.5Å. Currently I just tried once using phaser and refine the structure for 3 cycyles, I can get a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, especially those within helix or sheet, can be well described by 2Fo-Fc density. Is this primary result promising or not? 2. If it’s possible, what’s the general optimal procedure I should follow? Really thanks for any advice and suggestions! Zhihong

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Issue with SeMet residues
by Mark Wilson 07 Nov '13

07 Nov '13

Dear PHENIX users and developers, I am refining a structure containing SeMet residues at atomic (1.05 Å) resolution. The model was initially refined in Refmac5 and behaved as expected (i.e. reasonable R factors and agreement with maps). I've used this final Refmac5 model as a starting point for refinement in PHENIX 1.8.4 with standard settings, as below. phenix.refine data.mtz \ refinement.input.xray_data.labels="F, SIGF" model.pdb \ main.use_experimental_phases=false \ refinement.main.number_of_macro_cycles=5 output.prefix="model_refine"> phenix_refine_1.log Strangely, the conformations of the MSE residues are changed quite significantly after refinement in PHENIX, departing both from electron density and from the prior model. Any help would be appreciated. Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilson13(a)unl.edu

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Issues using reference model with Autobuild
by Dan McNamara 07 Nov '13

07 Nov '13

Hello all, I'm hoping to troubleshoot a problem with autobuild and the use of a reference model. Autobuild has been always failing once it gets to the initial refinement step and attempts to add reference model restraints. I've included my autobuild parameterization. Refinement is where it dies and the contents of LAST.LOG and my error log file are below too. I'm not sure how to get the reference model file location properly interpreted. I even tried manually moving the reference PDB into the AutoBuild_run_X_/ and TEMP0/ folders as they're generated to no avail. Best, Dan phenix.autobuild \ data=/home/.../xyz.mtz \ refinement_file=/home/.../xyz.mtz \ input_refinement_labels="FP SIGFP FreeRflag" \ model=/home/.../xyz.pdb \ seq_file=/home/.../xyz.dat \ s_annealing=False \ rebuild_in_place=True \ n_cycle_rebuild_max=3 \ refine_eff_file=/home/.../xyz.eff \ refine_with_ncs=True \ model_building.reference_model=/home/.../def.pdb \ #I have also tried locations like ../def.pdb and def.pdb that give Errno 21 or Errno 2 below nproc=200 \ general.run_command="/home/.../xyz.sh" AutoBuild_run_1_/LAST.LOG: ================== Extract refinement strategy and selections ================= Refinement flags and selection counts: individual_sites = True (30132 atoms) torsion_angles = False (0 atoms) rigid_body = True (30120 atoms in 24 groups) individual_adp = False (iso = 0 aniso = 0) group_adp = True (30132 atoms in 4092 groups) tls = True (30120 atoms in 147 groups) occupancies = True (12 atoms) group_anomalous = False n_use = 30132 n_use_u_both = 30120 n_use_u_iso = 12 n_use_u_aniso = 0 n_grad_site = 0 n_grad_u_iso = 0 n_grad_u_aniso = 0 n_grad_occupancy = 0 n_grad_fp = 0 n_grad_fdp = 0 total number of scatterers = 30132 *** Adding Reference Model Restraints *** errors.txt: Sorry: ******************************************************************************** Failed to carry out AutoBuild_build_cycle: Initial phenix refinement of AutoBuild_run_1_/TEMP0/NO_LIG_NO_WAT_xyz_ed.pdb with aniso_refinement_data_PHX.mtz failed...quitting... /home/.../AutoBuild_run_1_/LAST.LOG may have additional information... Cannot open file for reading: "/" [Errno 21] Is a directory: '/' ******************************************************************************** Sorry: ******************************************************************************** Failed to carry out AutoBuild_build_cycle: Initial phenix refinement of AutoBuild_run_2_/TEMP0/NO_LIG_NO_WAT_xyz_ed.pdb with aniso_refinement_data_PHX.mtz failed...quitting... /home/.../AutoBuild_run_2_/LAST.LOG may have additional information... Cannot open file for reading: "." [Errno 21] Is a directory: '.' ******************************************************************************** Sorry: ******************************************************************************** Failed to carry out AutoBuild_build_cycle: Initial phenix refinement of AutoBuild_run_3_/TEMP0/NO_LIG_NO_WAT_xyz_ed.pdb with aniso_refinement_data_PHX.mtz failed...quitting... /home/.../AutoBuild_run_3_/LAST.LOG may have additional information... Cannot open file for reading: "d" [Errno 2] No such file or directory: 'd' ********************************************************************************

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Sigmas of F+ and F- equal in _data file
by Tanner, John J. 07 Nov '13

07 Nov '13

I have a question about the _data.mtz file that phenix.refine outputs (version 1.8-1069). It appears that sigmaF+ equals sigmaF- for each reflection in the _data.mtz file, although they were unequal in the input .mtz file. You can compare the input mtz and the output _data.mtz file by viewing these links: http://faculty.missouri.edu/~tannerjj/input.jpeg http://faculty.missouri.edu/~tannerjj/samesigma.jpeg Thanks. Jack Tanner John J. Tanner Professor of Biochemistry and Chemistry University of Missouri-Columbia 125 Chemistry Building Columbia, MO 65211 Phone: 573-884-1280 Fax: 573-882-2754 Email: tannerjj(a)missouri.edu<mailto:[email protected]> http://faculty.missouri.edu/~tannerjj/tannergroup/tanner.html

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real-space refinement
by "Weiergräber, Oliver H." 06 Nov '13

06 Nov '13

Hello, beginning with phenix 1.8.3, the individual_sites_real_space option will not run globally if, e.g., R < 0.25. Is there a way to force the original behaviour? Regards, Oliver ================================================ PD Dr. Oliver H. Weiergräber Institute of Complex Systems ICS-6: Structural Biochemistry Tel.: +49 2461 61-2028 Fax: +49 2461 61-9540 ================================================ ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------ Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher Geschaeftsfuehrung: Prof. Dr. Achim Bachem (Vorsitzender), Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt, Prof. Dr. Sebastian M. Schmidt ------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------

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Is the protein-ligand complex structure model right??
by Wei Shi 06 Nov '13

06 Nov '13

Hi all, I am trying to solve a protein-ligand complex structure. The resolution of the data is 2.8Å. The molecular replacement solution is found in space group P21 21 21. The current best model: start final ------------------------------------------------ R-work: 0.2836 0.2484 R-free: 0.3236 0.3066 RMS(angles): 0.60 0.63 RMS(bonds): 0.002 0.002 ==============MolProbity validation ============== Ramachandran outliers: 2.4% (Goal: < 0.2%) Ramachandran favored: 89.2% (Goal: > 98%) Rotamer outliers: 4.7% (Goal: 1%) C-beta outliers: 0 (Goal: 0) Clashscore: 10.89 Overall score: 2.64 Four ligands are placed for every dimer of the protein. And we are not sure whether we put the ligands right, since first it's thought that only two ligands bind every dimer of the protein. I created a SA omit map (composite). The density for the most of the N-terminal DNA binding domain is very bad, and the density for C-terminal ligand binding domain is much better. And, I could see density for all 4 ligands. Does this necessarily mean that the model for the N-terminal DNA binding domain is not right or should I try other types of omit map (simple, refined or iterative building)? Also, I am wondering what's the difference between different types of omit map (simple, refined, iterative building) and whether it's the most reliable method to tell whether the structure model is right. So, I am not sure whether the structure model I have is correct. It's suspected that the molecular solution is in the wrong space group or the data is twinned and only two ligands bind every dimer of the protein......But, I am not sure what I should check or what to do to improve the model. Thank you so much! Best, Wei

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Is the protein-ligand complex structure model right??
by Wei Shi 06 Nov '13

06 Nov '13

Hi all, I am trying to solve a protein-ligand complex structure. The resolution of the data is 2.8Å. The molecular replacement solution is found in space group P21 21 21. The current best model: start final ------------------------------------------------ R-work: 0.2836 0.2484 R-free: 0.3236 0.3066 RMS(angles): 0.60 0.63 RMS(bonds): 0.002 0.002 ==============MolProbity validation ============== Ramachandran outliers: 2.4% (Goal: < 0.2%) Ramachandran favored: 89.2% (Goal: > 98%) Rotamer outliers: 4.7% (Goal: 1%) C-beta outliers: 0 (Goal: 0) Clashscore: 10.89 Overall score: 2.64 Four ligands are placed for every dimer of the protein. And we are not sure whether we put the ligands right, since first it's thought that only two ligands bind every dimer of the protein. I created a SA omit map (composite). The density for the most of the N-terminal DNA binding domain is very bad, and the density for C-terminal ligand binding domain is much better. And, I could see density for all 4 ligands. Does this necessarily mean that the model for the N-terminal DNA binding domain is not right or should I try other types of omit map (simple, refined or iterative building)? Also, I am wondering what's the difference between different types of omit map (simple, refined, iterative building) and whether it's the most reliable method to tell whether the structure model is right. So, I am not sure whether the structure model I have is correct. It's suspected that the molecular solution is in the wrong space group or the data is twinned and only two ligands bind every dimer of the protein......But, I am not sure what I should check or what to do to improve the model. Thank you so much! Best, Wei

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Green density for ligands in places where the ligand is in density
by Wei Shi 06 Nov '13

06 Nov '13

Hi guys, I am working with a protein-ligand complex structure. I generated the ligand pdb and cif with Phenix.eLBOW. Then, I fit the ligand to density (green or continuous blue denisty) in Coot. Then, I did a refinement of the protein-ligand complex model in Phenix. I opened the refinement results in Coot, I don't know why in some places where the ligand is in density, it always shows green density in Coot... And, from the pdb file, the occupancy for all ligands are 0? Do any of you happens to know whether this is unusual and what might go wrong? Thank you so much! Best, Wei

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Re: [phenixbb] Restraints generation for TaBr
by Rebecca Oot 05 Nov '13

05 Nov '13

Hi Nigel, Thanks so much for the response, the information and for the files. Best, Rebecca

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Re: [phenixbb] Restraints generation for TaBr
by Rebecca Oot 05 Nov '13

05 Nov '13

Hi Anshul, Thanks very much for the files--much appreciated!! Hope all is well. Rebecca

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Restraints generation for TaBr
by Rebecca Oot 05 Nov '13

05 Nov '13

Hello all, I am trying to create a restraints file for hexatantlum dodecabromide (TaBr or TBR) and using Elbow, I get an error message saying that the program does not deal with heavy atom clusters. Does anyone have any advice on which program would be suitable to use? Thanks very much in advance Rebecca

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Re: [phenixbb] Phenix.Rosetta_refine on Ubuntu 12.04 LTS
by Jan Gebauer 05 Nov '13

05 Nov '13

Hi David, hi all, I now managed to at least get a test refinement working. What I mostly changed was the Ubuntu version. I have no idea why, but I managed to get it to work on Ubuntu 10.04. In principle I installed 10.04 freshly in a virtualbox and from their it worked rather straight-forward. Here is how I proceeded in detail for those of you who are interested: 1) Setting up a Virtualbox 4.3.2 r90405 on Ubuntu 12.04.3. 2) Installing the 64bit version of Ubuntu 10.04 in a virtualbox (4 processors / 90% speed cap / 4GB Ram /VT-x enabled) 3) Install VBoxAdditions 4) Update ubuntu (sudo apt-get update) 5) Installing g++ and zlib library (sudo apt-get install g++ zlib1g-dev) 6) Installing binary version of phenix-dev-1525 to /usr/local/science/phenix-dev-1525 (sudo ./install --prefix=/usr/local/science) 7) Unpack rosetta_2013_wk42_bundle to /usr/local/science/rosetta_2013_wk42_bundle 8) set rossetta variables by inserting them at the start of /usr/local/science/pheninx-dev-1525/phenix_env.sh (just in line 3ff): export PHENIX_ROSETTA_PATH=/usr/local/science/rosetta_2013wk42_bundle export ROSETTA3_DB=/usr/local/science/rosetta_2013wk42_bundle/main/database export PATH=$PATH:/usr/local/science/rosetta_2013wk42_bundle/main/source/bin 9) assign ownership to active user (sudo chown gebauer:gebauer /usr/local/science/ -R) 10) Compile it (phenix.build_phenix_interface nproc=4) It should run without problems Testing: 1) Create a new directory (mkdir test && cd test) 2) Download testset (suggested by Nat: phenix.fetch_pdb --mtz 1yjp) 3) Run rosetta_refine (phenix.rosetta_refine 1yjp.pdb 1yjp.mtz nproc=4) In my case rFree dropped from 18.21 to 14.90. It's not a low resolution dataset, but it show that it general works and lowRes would potentially take to long for a mere testing My real data are still being processed so I have no idea how well it will works... only thing I found out already: 4.5 GB RAM for 4 processes calculating ~500Aa via rosetta seems not to be enough. I thereby reduced my "real run" to only one processor... Regards, Jan Am 01.11.2013 08:39, schrieb David Goldstone: > Hi Jan, > > I followed the same procedure. The only differences being that I have > installed it to my home directory to keep it separate from our > standard install of phenix and rosetta and I run the phenix_env.sh > separately from the exports. I had to remove our default init.sh > script from my .bashrc (this is our setup script for all programs ccp4 > etc) and now manually source a different version to ensure it points > to the correct version of phenix. > > I originally had trouble with the python shared libraries but Nat has > fixed this by building them into the binary installer. > > Sorry I can't be more help > > David > > > ---- > Dr David Goldstone > Senior Lecturer > University of Auckland > New Zealand > Ph +64 9 3737599 x84607 <tel:+64%209%203737599;84607> > > On 1/11/2013, at 8:30 PM, Jan Gebauer <jan.gebauer(a)uni-koeln.de > <mailto:[email protected]>> wrote: > >> Dear David, >> >> thanks for your remark. Indeed my paths are wrong in the mail; >> however, this is due to lazy typing... I do set the Rosetta_DB3 path >> corretly... >> I'm still interested to know, how you managed to install build it. >> Have you done the same procedure as described by me? (only with >> different releases?) >> Or did you do something differently? >> >> Regards, >> Jan >> >> Am Donnerstag, 31. Oktober 2013 23:11:42 schrieb David Goldstone: >>> Dear Jan, >>> >>> I have recently installed phenix with rosetta refine on Ubuntu 10.04 >>> using phenix-dev-1515 and rosetta_wk40_bundle. >>> >>> This install also appears to run on an Ubuntu 12 install. >>> >>> I think you export commands are not correct mine are as follows >>> >>> export >>> PHENIX_ROSETTA_PATH=/mnt/home/dgol002/programs/rosetta_2013wk40_bundle >>> export >>> ROSETTA3_DB=/mnt/home/dgol002/programs/rosetta_2013wk40_bundle/main/database >>> export >>> PATH=$PATH:/mnt/home/dgol002/programs/rosetta_2013wk40_bundle/main/source/bin >>> >>> ie you are exporting ROSETTA_BIN twice rather than ROSETTA3_DB. >>> >>> Hope this helps >>> >>> David >>> >>> >>> >>> On 1/11/2013 4:48 a.m., Jan Gebauer wrote: >>>> Dear all, >>>> >>>> I'm trying to get rosetta_refine working on ubuntu 12.04 LTS. >>>> >>>> This was the (latest) way I tried it: >>>> >>>> 1) Downloaded phenix-dev-1523 binary & installed it via installer in >>>> /usr/local/science/phenix4rosetta/phenix-dev-1523 >>>> 2) Downloaded rosetta 2031wk42 and extracted it in >>>> /usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle >>>> 3) Inserted into phenix_env.sh >>>> export >>>> ROSETTA_BIN=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/main/source/bin >>>> export >>>> PHENIX_ROSETTA_PATH=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/ >>>> export >>>> ROSETTA_BIN=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/main/database >>>> 4) sources phenix_env.sh >>>> 5) build the interface via >>>> rosetta.build_phenix_interface nproc=4 >>>> 6) exceute the refinement via phenix.rosetta_refine phasermodel.pdb >>>> data.mtz nproc=4 >>>> >>>> The programm runs fine, up to the point where it should generate >>>> models via rosetta, it then stops and crashes. >>>> I have attached some logfiles >>>> >>>> Has anyone managed to run rosetta_refine on Ubuntu? Which phenix and >>>> rosetta versions did you use? Ist there anything wrong on my above >>>> mentioned build protocol? >>>> >>>> I'm thankful for any help. >>>> >>>> Regards, >>>> Jan Gebauer >>>> >>>> >>>> >>>> -- >>>> Dr. Jan Gebauer >>>> AG Prof. Baumann >>>> Institut für Biochemie / Uni-Köln >>>> Otto-Fischer-Str. 12-14 / 50674 Köln >>>> Fon: +49 (221) 470 3212 >>>> Fax: +49 (221) 470 5066 >>>> >>>> http://px.uni-koeln.de/ >>>> >>>> >>>> _______________________________________________ >>>> phenixbb mailing list >>>> phenixbb(a)phenix-online.org <mailto:[email protected]> >>>> http://phenix-online.org/mailman/listinfo/phenixbb >>> >>> >>> -- >>> Dr David Goldstone >>> Senior Lecturer & Rutherford Discovery Fellow >>> Structural Biology >>> University of Auckland >>> New Zealand >>> Ph +64 9 3737599 x84607 -- Dr. Jan Gebauer AG Prof. Baumann Institut für Biochemie / Uni-Köln Otto-Fischer-Str. 12-14 / 50674 Köln Fon: +49 (221) 470 3212 Fax: +49 (221) 470 5066 http://px.uni-koeln.de/

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Post-doctoral Fellowships at the University of Texas
by Gabrielle Rudenko 04 Nov '13

04 Nov '13

*Macromolecular Crystallography, Biophysics and Biochemistry* Two post-doctoral fellowship positions are available at the University of Texas Medical Branch (UTMB) located in the greater Houston area in the research team of Dr. Gabrielle Rudenko. We study molecules that mediate the formation, maintenance and plasticity of synapses, the contact and communication point between neurons. Our proteins of interest organize and regulate protein interaction networks in the synaptic cleft and impact how neurons communicate with each other at synapses. Many of the proteins we study are implicated in neurological disease and serve as potential novel therapeutic targets. We are looking for highly motivated, enthusiastic, hard working individuals who are interested in combining the fields of structural biology and neuroscience. Research projects involve protein over-expression and purification, x-ray crystallography, biochemical and biophysical techniques, proteomics, chemical biology and in collaboration electron microscopy and NMR. Our laboratory is joining the endowed Sealy Center for Structural Biology and Molecular Biophysics and the Department of Pharmacology/Toxicology at UTMB. The Sealy Center provides access to exceptional facilities, training and experimental assistance. *X-ray facilities* include a high-brilliance FR-E Superbright x-ray generator, a Rigaku R-AXIS-IV dual 30cm Imaging Plate detector, a Bruker SMART 2K CCD and a Rigaku BioSAXS-1000 2D-Kratky camera (for solution scattering data). *Biophysics facilities* include equipment for analytical ultracentrifugation, fluorescence, MALDI-TOF mass spectrometry, dynamic light scattering, surface plasmon resonance, titration micro-calorimetry, and high throughput screening with the ThermoFluor HTS apparatus. In addition, the *Electron Microscopy facilities*house three modern JEOL cryo-electron microscopes (a 200 keV JEOL 2200FS cryo-EM with in-column electron energy filter and field emission gun, a 200 keV JEOL 2100 EM, and a 120 keV JEM1400 microscope) and the *NMR facilities*include three NMR spectrometers (Bruker Avance III 800, 750 and 600 MHz with cryoprobes). See http://www.scsb.utmb.edu/resources/ UTMB is part of the Texas Medical Center, the world’s largest medical center located in greater Houston. UTMB is situated on Galveston Island offering a pleasant subtropical climate and miles of relaxing beaches. UTMB is located within commuting distance of Houston, the fourth largest city in the United States and a leader in the arts, education, and health care. *Qualifications:* Applicants should have received a Ph.D. degree in a field relevant to structural biology in the past three years and have a strong publication record. Applicants with experience in cloning, bacterial/insect cell culture, protein purification and crystallization, and macromolecular structure determination are preferred. However, recent graduates with a strong background in protein over-expression and purification, and an exceptional interest in learning crystallography are also encouraged to apply. Good written and oral communication skills in English are essential as well as the ability and desire to work as part of a highly motivated, collaborative team. To apply please e-mail the following to *rudenkolab.application(a)gmail.com*<rudenkolab.application(a)gmail.com>: CV, contact information for three references (address, phone and e-mail), a list of your experimental expertise and skills relevant to this position, and a research statement describing your past research experience and future goals (1-2 pages). Visa sponsorship is available for non-US nationals. For questions please contact Dr. Gabrielle Rudenko at <https://web.mail.umich.edu/blue/imp/message.php?mailbox=INBOX&index=111546> *rudenko(a)umich.edu* <rudenkolab.application(a)gmail.com>. For more information please visit http://www.lsi.umich.edu/facultyresearch/labs/rudenko. The University of Texas is a non-discriminatory, affirmative action employer. -- Gabrielle Rudenko PhD Life Sciences Institute and the Dept. of Pharmacology University of Michigan Medical School 210 Washtenaw Ave Rm 3163A Ann Arbor, Michigan 48109-2216 734 - 615 9323 rudenko(a)umich.edu www.lsi.umich.edu/facultyresearch/labs/rudenko

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Re: [phenixbb] Appropriate number of reflections for FreeR
by Edward Berry 03 Nov '13

03 Nov '13

Looking at it from the other side- suppose we say for a robust refinement we need a certain number of reflections- say 4 times the number of atoms, maybe less, I don't know. Any more than that is not really going to affect the structure, So if you have high resolution you can afford to use a large percentage of free reflections, and the cross-validation and maximum liklihood will go really well, even with thin shells. On the other hand at low resolution, well, sorry, you really can't spare any reflections for cross-validation. (I have no idea whether this makes sense or not, but it would be another way of looking at it). >>> Mark van Raaij 11/01/13 7:39 PM >>> the limit of 2000 reflections I guess is just because it would be a waste to "throw away" more reflections for refinement, once the statistical minima for calculating a reliable Rfree have been met. I.e. if you have 100.000 reflections, it would be a waste to use 5 or 10% of the reflections instead of just 2%. I'd rather use as many as possible reflections for refinement. On 31 Oct 2013, at 20:21, Pavel Afonine wrote: > Hi Joe, > > flags should be selected such that there is enough of them in each relatively thin resolution shell (thin enough so ML target parameters can be considered constant in each such shell). Lower end is about 50 reflections per shell. All in all this usually translates into about 10% overall. > > Yes, there is a limit parameter set to 2000 by default. I don't know what's the rationale for having it, may be someone can explain. > > Pavel > > On 10/31/13 12:10 PM, Joseph Noel wrote: >> Hi All. I think I have asked this before but forgot. Old age. What is >> the appropriate number / percentage of reflections to flag for a >> statistically appropriate Free-R calculation? If I am correct, the >> reflection file editor in Phenix chooses by default either 10% of the >> measured reflections or 2000 whichever comes first. >> ______________________________________________________________________________________ >> Joseph P. Noel, Ph.D. >> Arthur and Julie Woodrow Chair >> 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 >> >> Publications & Citations: >> http://scholar.google.com/citations?user=xiL1lscAAAAJ >> >> Homepage Salk: http://www.salk.edu/faculty/noel.html >> Homepage HHMI: http://hhmi.org/research/investigators/noel.html >> ______________________________________________________________________________________ >> >> >> >> _______________________________________________ >> phenixbb mailing list >> phenixbb(a)phenix-online.org >> http://phenix-online.org/mailman/listinfo/phenixbb >> > _______________________________________________ > phenixbb mailing list > phenixbb(a)phenix-online.org > http://phenix-online.org/mailman/listinfo/phenixbb _______________________________________________ phenixbb mailing list phenixbb(a)phenix-online.org http://phenix-online.org/mailman/listinfo/phenixbb

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Appropriate number of reflections for FreeR
by Joseph Noel 03 Nov '13

03 Nov '13

Hi All. I think I have asked this before but forgot. Old age. What is the appropriate number / percentage of reflections to flag for a statistically appropriate Free-R calculation? If I am correct, the reflection file editor in Phenix chooses by default either 10% of the measured reflections or 2000 whichever comes first. ______________________________________________________________________________________ Joseph P. Noel, Ph.D. Arthur and Julie Woodrow Chair 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 Publications & Citations: http://scholar.google.com/citations?user=xiL1lscAAAAJ Homepage Salk: http://www.salk.edu/faculty/noel.html Homepage HHMI: http://hhmi.org/research/investigators/noel.html ______________________________________________________________________________________

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Re: [phenixbb] Appropriate number of reflections for FreeR
by Pavel Afonine 03 Nov '13

03 Nov '13

Hi Rongjin, it's not really waste: test reflections are used not only in Rfree calculations, but in ML refinement target parameters estimations, so more test reflections you use better these estimations are, and so better refinement target is. I still believe that 10% is a good number, and any capping the amount of test reflections is NOT necessary: it's not about getting a statistically meaningful amount of them overall, but rather it's about getting the right spread of them across the whole resolution range, and the right amount across all of the thin slices of resolution. I imagine by capping them at a particular number you may end up having a couple of test reflections per certain bin, which isn't great. Pavel On 11/1/13 9:18 PM, Rongjin Guan wrote: > Thank you, Nat and Pavel. I just came back and saw replies from both of > you. Thanks a lot. > I have an impression that free_R set has max 2000 reflections, and more > importantly, each resolution shell has at least > 50 reflections, so I feel >4500 in the free_R set / 150 each shell kind > of luxury ("waste"), and if I have more reflections in the refinement > set, I could have better data/parameters ratio. But this may not make > much difference. > Best, and have a great weekend. > Rongjin > > > On Fri, Nov 1, 2013 at 3:18 PM, Nathaniel Echols <nechols(a)lbl.gov > <mailto:[email protected]>> wrote: > > On Fri, Nov 1, 2013 at 12:13 PM, rjguan <rjguan(a)gmail.com > <mailto:[email protected]>> wrote: > > I solved a structure with 2.7 A Se-Met data set, with 10% > reflections in free_R set. > > Now I have 2.0 A native data set, and extended free_R to 2.0A. > Now I have >4500 reflections > > in free_R set, each resolution shell has >150 reflections. Kind > of too much, right? > > I don't think it's necessarily too much, but it is probably more > than you need. > > > > What is the best way to reduce the number of reflections in the > free_R set, say, to 5%? > > I already built and refined model at 2.7 A, but do not want to > redo autobuild. > > Use the reflection file editor in the GUI - click "More options" in > the section for R-free flags, and check the box "Adjust test set > size to specified fraction". > > > Another question: now I have 2.0A data set, shall I use phases > from 2.7 A data in refinement? > > I compared, without using the phases I got lower R/R_free (about > 1% lower). > > is this because the 2.0 A data is more accurate than the 2.7 A > phases, and I should continue > > to refine at 2.0A without the phases from 2.7 A data? > > It's difficult to reach a conclusion by comparing R-factors alone; > maybe you simply need to run more cycles of refinement with phases > to get the same result. Alternately, if the crystals aren't truly > isomorphous, the phases may be inappropriate for the native > structure. But I think with 2.0Å data and a complete model, it is > perfectly valid to use the amplitudes-only ML refinement target. > > -Nat > >

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Phenix.Rosetta_refine on Ubuntu 12.04 LTS
by Jan Gebauer 31 Oct '13

31 Oct '13

Dear all, I'm trying to get rosetta_refine working on ubuntu 12.04 LTS. This was the (latest) way I tried it: 1) Downloaded phenix-dev-1523 binary & installed it via installer in /usr/local/science/phenix4rosetta/phenix-dev-1523 2) Downloaded rosetta 2031wk42 and extracted it in /usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle 3) Inserted into phenix_env.sh export ROSETTA_BIN=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/main/source/bin export PHENIX_ROSETTA_PATH=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/ export ROSETTA_BIN=/usr/local/science/phenix4rosetta/rosetta_2013wk42_bundle/main/database 4) sources phenix_env.sh 5) build the interface via rosetta.build_phenix_interface nproc=4 6) exceute the refinement via phenix.rosetta_refine phasermodel.pdb data.mtz nproc=4 The programm runs fine, up to the point where it should generate models via rosetta, it then stops and crashes. I have attached some logfiles Has anyone managed to run rosetta_refine on Ubuntu? Which phenix and rosetta versions did you use? Ist there anything wrong on my above mentioned build protocol? I'm thankful for any help. Regards, Jan Gebauer -- Dr. Jan Gebauer AG Prof. Baumann Institut für Biochemie / Uni-Köln Otto-Fischer-Str. 12-14 / 50674 Köln Fon: +49 (221) 470 3212 Fax: +49 (221) 470 5066 http://px.uni-koeln.de/

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warning: do not upgrade Mac OS yet!
by Nathaniel Echols 30 Oct '13

30 Oct '13

There is some kind of strange issue with wxPython on the new Mac OS ("Mavericks"), which for at least the 1.8.4 version prevents Phenix application windows (but not the main GUI) from opening. It is also printing out an extraordinary number of warning messages about fonts to the console. I'm hoping there is a simple solution (which seems likely, since 1.8.2 is mostly okay despite using the same version of wxPython), but until we figure out what is causing this, I recommend leaving your Macs unmolested if you want to use the Phenix GUI. Updates to follow if-and-when I fix this. thanks, Nat

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Using phenix_rosetta_refine
by Jan Gebauer 29 Oct '13

29 Oct '13

Dear all, I tried to install phenix.rosetta_refine for a recently solved structure, which only diffracts up to 3.5-4.0 Angstrom (anisotropic). However, I was unable to get the programs correctly set up. I first tried to compile phenix 1.8.4-1496 from source according to this documentation : http://www.phenix-online.org/version_docs/dev-1500/rosetta_refine.htm. The build process failed during GTK2 building, without an obvious explanation (error code 1, no missing libraries ...) I then realised that the developer version are potentially more suitable, as they seem to be prepared for this. I installed the binary from 1523 and tried to start the building process from there with rosetta.build_phenix_interface nproc=X Unfortunatelly Rosetta 3.5 seems to have a different folder structure. There is no main subfolder, instead there are "rosetta_sources", "rosetta_tools" and so one. After a fast "cp rosetta_sources main/source/" it worked a bit but still exited with: scons: *** Error converting option: extras Invalid value(s) for option: python File ..... /setup.py, line 72, in setup_build_options I have the following questions: 1) Am I right to assume that I do NOT have to recompile the dev 1523 (1520 states "Python shared library for linux) on Linux? 2) Which rosetta version do I have to use? Does 3.5 work? There is no 3.6 on rosettacommons... (or is it hidden?) Regards, Jan -- Dr. Jan Gebauer AG Prof. Baumann Institut für Biochemie / Uni-Köln Otto-Fischer-Str. 12-14 / 50674 Köln Fon: +49 (221) 470 3212 Fax: +49 (221) 470 5066 http://px.uni-koeln.de/

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Need help with atom selection for anisotropic refinement
by Dr. Mark Mayer 28 Oct '13

28 Oct '13

Hi, I'm trying to do a mixed individual isotropic anisotropic refinement b factor refinement and can't get my atom selections to be accepted by phenix.refine. For the atoms I want to do isotropic refinement I used optional (element H or element D or resname HOH or chain E or chain F or chain A or chain B) for the anisotropic selection I used chain A and chain B and not (element H or element D or resname HOH or chain E or chain F or chain A resseq 5:11 or chain A resseq 66:72 or chain A resseq 117:138 or chain A resseq 152:184 or chain A resseq 196:210 or chain A resseq 249:251 or chain B resseq 3:6 or chain B resseq 66:72 or chain B resseq 166:210 or chain B resseq 249:251) I've tried various combuinations, but so far all have given the error message: Selection string results in empty selection. Thanks for your help Mark --

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Alternative Conformation, Restraints
by Moritz Machelett 27 Oct '13

27 Oct '13

Hello everyone, I am in the final rounds of refining my protein at high resolution and currently trying to model an alternative conformation of a Chl /a/-phytol chain. I inserted a second phytol chain with coot - worked out well so far (alternate chain identifiers A and B for both phytol chains). After using phenix.refine, however, the occupancy of the second phytol chain was refined to 0. When splitting the whole Chl molecule the occupancy refines to sensible values (0.34 and 0.66). We would prefer to only model the phytol chain in alternate conformation as the macrocycle superpose perfectly. Any suggestions regarding the occupancy problem? Another issue concerns restraints. We tried to turn of specific nonbonded restraints and found a phenixbb message from 2007: http://www.phenix-online.org/pipermail/phenixbb/2007-September/011734.html Are there any news/updates on this matter? Any help appreciated! Thanks, Moritz -- Moritz M. Machelett Ruhr University Bochum Protein Crystallography, Department of Biophysics, ND04/396 44780 Bochum Tel: +49-(0)234/32-25754

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fem map
by nih\mayerm 27 Oct '13

27 Oct '13

Hi, I'm trying to make a fem amp using phenix-1.8.4-1496 using my refined structure and mtz with map coefficients as input: phenix.development.fem 3222_Glu_refine_78.mtz 3222_Glu_refine_78.pdb coot 0.7.0-1 fails to generate a map when fem.mtz is opened, either in auto or manual mode mtzdmp for fem.mtz gives: Col Sort Min Max Num % Mean Mean Resolution Type Column num order Missing complete abs. Low High label 1 NONE -37 35 0 100.00 -5.0 14.2 61.69 1.21 H H 2 NONE 0 101 0 100.00 38.0 38.0 61.69 1.21 H K 3 NONE 0 44 0 100.00 16.8 16.8 61.69 1.21 H L 4 NONE 0.0 1796.7 25825 84.81 84.67 84.67 31.87 1.21 F 2mFoDFc 5 NONE -180.0 180.0 25825 84.81 1.07 90.28 31.87 1.21 P PHI2mFoDFc 6 NONE 0.0 2750.2 0 100.00 144.76 144.76 61.69 1.21 F 2mFoDFc_FilSharp 7 NONE -180.0 180.0 0 100.00 1.26 90.34 61.69 1.21 P PHI2mFoDFc_FilSharp 8 NONE 0.2 24426.8 0 100.00 235.27 235.27 61.69 1.21 F KICK 9 NONE -180.0 180.0 0 100.00 0.02 89.63 61.69 1.21 P PHIKICK 10 NONE 0.2 9283.0 0 100.00 132.01 132.01 61.69 1.21 F FEM 11 NONE -180.0 180.0 0 100.00 0.23 90.34 61.69 1.21 P PHIFEM

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Using experimental phases in refinement
by Joseph Noel 26 Oct '13

26 Oct '13

Hi Folks, I am wondering if anyone can recommend some good reference(s) of structure refinements that employed experimental phases. We are attempting to collect experimental phases for a series of structures for which we have refined the structure already. I assume we can easily use initial model phases (space group doesn't change) to locate anomalous scatters and calculate experimental phases from the elucidation of the heavy atom arrangement. Most of the structures range from 1.7 to 2.4 A resolution. Would also appreciate any advice on the most efficient and accurate set of steps to accomplish this. If anyone thinks that this will not add much to the refinement of these structures would also appreciate some critiques. The protein in question crystallizes with 68% solvent content and one monomer per assymetric unit. We are employing ensemble refinement as the protein in question displays local conformational changes depending on the type of ligand(s) bound and also pH and similar but distinct crystallization conditions. We are carrying this out on ~400 mutants to do a very large scale comparison. Sorry for the long email but since we want to try and understand even minor conformational changes I thought phased-refinement would be much more powerful then refining just against the Fs. Thanks again in advance, Joe ______________________________________________________________________________________ Joseph P. Noel, Ph.D. Arthur and Julie Woodrow Chair 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 Publications & Citations: http://scholar.google.com/citations?user=xiL1lscAAAAJ Homepage Salk: http://www.salk.edu/faculty/noel.html Homepage HHMI: http://hhmi.org/research/investigators/noel.html ______________________________________________________________________________________

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protein-ligand complex structure
by Wei Shi 26 Oct '13

26 Oct '13

Hi all, I am working on a structure of protein-ligand complex. Four ligands are placed for the dimer protein and the density for the two ligands of the first monomer is better than the density for the other two ligands of the second monomer. Ligand is moved to fit density better in Coot (and for two ligands of the first monomer, they fits the density almost perfectly), but after a refinement in phenix (default settings + NCS restraints + Secondary structural restraints + Optimize X-ray/stereochemistry weight +Optimize X-ray/ADP weight), some part of the ligand which fits density good before moved out of the density again...Besides, it always shows green density in Coot for the ligand region even in places where the ligand is in density... Any suggestions or ideas about how to fit the ligand better and why the density for ligands of the second monomer is worse than that for the first monomer and why the ligands would move out of density after refinement? Is it because the protein model is not good enough to get the ligand density good? There is a conformational change upon ligand binding, and I rebuilt some part of the protein manually, and the density for a region of about 30 residues is not very good, and I tried to mutate those to alanies and refine, but it didn't help me see the density better... Any suggestions or ideas on how to improve this protein-complex structural model? Thank you so much! The statistics for the current best model is as follows, and the resolution of the dataset is 2.8Å. start final ------------------------------ ---------------- R-work: 0.3359 0.2993 R-free: 0.3619 0.3558 RMS(angles): 1.03 1.55 RMS(bonds): 0.006 0.007 MolProbity validation Ramachandran outliers: 4.7% (Goal: < 0.2%) Ramachandran favored: 85.3% (Goal: > 98%) Rotamer outliers: 4.5% (Goal: 1%) C-beta outliers: 0 (Goal: 0) Clashscore: 7.43 Overall score: 2.56 Thank you so much! Best, Wei

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