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Help needs: SAD-phasing for Cobalt ion
by YoungJin 10 Dec '10

10 Dec '10

Dear helpers in phenix, I have anomalous data (Co; wavelength: 1.609A) that I want to use its Phase information from SAD-phasing method (autosol). As I have a native data (from nomal wavelength ~1A) with higher resolution (~2A) than SAD data (~2.5A), I wanted to apply phase information to this native data for further refinement: My goals were 1. I want to confirm where is my heavy metal is; 2. I want to refine further using native data as has higher resolution. At first, when I did autosol, it didn't give me perfect result. So far what I tired is as following: 'SAD peak' from imosflm generated mtz file (scaled), 'Phases' from first refmac generated mtz file from the native data, and 'MIR native' from the native data mtz file (imosflm and scaled). FOM: 0.186; overall score was 10.75 +/- 12.33. FYI. I input 2 sites and result suggested 3 sites. Each molecule has one binding site of metal ion. I was using old Mac version:1.6.4-486 Anyway, I simply passed the result over Autobuild. It ran and gave me better statistics as following: R/Rfree=0.2734/0.3312; CC 0.76. However (I have two molecules/ASU) one of models was not great. But this time, anomalous density map well phased(?!) to 2Fo-Fc map and I could see - it was before only one heavy metal I clearly could see- 2 heavy metal signal from 2 molecules and those positions are well matched with native density maps. So I continued phenix.refine using Autobuild made 'expt_fobs_phase_freeR_flags_1.mtz'. The question is the statistics from anomalous data itself (it has slight dispcrepancy between anomalous map and 2Fo-Fc/Fo-Fc map due to phase problem) or even those from native data is better than my current model which came from native data set (SAD-phased as described above) with higher resolution. I think one of main reasons is one of molecule has a lid that does not perfectly fit into current density map. However, in terms of heavy metal position and the active site is better from current map (Autobuild generated model). FYI: current model has R/Rfree=0.21/0.27(best was 0.21/0.26); 0.19/0.24 (for native data only used model) .... Am I too concerning for statistics or is there something I am missing? Now my questions are as following plus any suggestions are welcome: 1. For SAD-phasing, could you tell me which is best way to do so with my case (one data from the edge of heavy metal (lower resolution) and native data (higher resolution) or where is the point I can convince. Also I tried to use DANO/SIGDANO column (from hkl2000 and ccp4) but seems like it (Autosol) doesn't take them into account. Could you tell me how can I improve phase by using anomalous data? 2. In case of Co, does anyone have experience about coordination numbers? I thought it should have 6 (or 4) coordination numbers. The question what I have now is about the numbers and their lengths ... I though it should be within 2A, but not true for my current models (And somehow distorted octahedron or else with 2.07-3.28A). I also searched PDB files, and found that many deviations from 1.98 to 2.82A (depending on corresponding atoms and resolutions: 3KOI, 3KON, 2DD5, and 2XJM). I would appreciate if you can give me any tips regarding this issue. Many thanks in advance, Young-Jin

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phenix.phaser ep_sad mode
by Bryan Lepore 09 Dec '10

09 Dec '10

dev-601 i found an ep_sad script on the phaser wiki^[1] that works ok^[2] for old versions. for the dev-601 phaser, i think it comes down to me not understanding this line : LLGComplete CRYStal insulin COMPLETE ON SCATtering ELEMent S i tried jumbling the commands/arguments around^[3], leading to syntax errors for each attempt (output excerpt below). if this "should work", i'll gladly return to the manual - or is there something unusual between versions, am i missing something, etc.... -Bryan [1]http://www.phaser.cimr.cam.ac.uk/index.php/Experimental_Phasing#Combined_MR_and_SAD_Phasing [2] seem not to write ATOM records in the .pdb when sites are in fact found. [3]http://www.phaser.cimr.cam.ac.uk/index.php/Keywords#LLGCOMPLETE output excerpt : MODE EP_SAD TITLe sad phasing insulin with best AutoMR solution HKLIn truncate.mtz CRYStal insulin DATAset sad LABIn F+=F(+) SIG+=SIGF(+) F-=F(-) SIG-=SIGF(-) ! LLGComplete CRYStal insulin COMPLETE ON SCATtering ELEMent S LLGComplete CRYStal  <B><FONT COLOR="#FF8800"> $TEXT:Warning: $$ Baubles Markup $$ ------------------------------------------------------------------------------------- SYNTAX ERROR: Use (optionally) COMPLETE TYPE SCATTERER ELEMENT CLUSTER SIGMA NCYC CLASH ANOM REAL MAPS or METHOD -------------------------------------------------------------------------------------

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phaser ep_sad zscore
by Bryan Lepore 09 Dec '10

09 Dec '10

dev-601 in phaser ep_sad mode, i tried ZSCORE SOLVED 0 to get more sites output, and i can see a peak of about Z=4 in the stdout, but no peaks are written to the .pdb am i applying ZSCORE wrong here? -Bryan

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reflection outliers detection in phenix.refine
by guanrj＠foxmail.com 08 Dec '10

08 Dec '10

Hi All I have a question regarding reflection outliers detection in phenix.refine: in my input reflection file, there are 24432 reflections, but only 22769 of them were used in refinement. I checked the log file and saw 1663 outliers. (below if from the log) basic_wilson_outliers = 14 extreme_wilson_outliers = 18 beamstop_shadow_outliers = 0 model_based_outliers = 1657 total = 1663 What is the "model_based_outliers"? What causes so many reflections rejected and not used in the refinement? Thanks Rongjin

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Multistart simulated annealing refinement
by Joseph Noel 08 Dec '10

08 Dec '10

Hi All, I'm sure this has already been addressed but is there a way through the PHENIX GUI to carry out multistart simulated annealing refinement either with different starting seeds (velocities) or temperatures automatically or does one have to start a fresh run each time? Thanks! Joe P.S. Similar procedure as described in the PNAS paper: "Multistart simulated annealing refinement of the crystal structure of the 70S ribosome" ___________________________________________________________ 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.fmodel or other utility: calculate HL's as well?
by Francis E Reyes 08 Dec '10

08 Dec '10

I love the phenix cmd line utilities, however is there one that'll calculate HLA/HLB/HLC/HLD given FModel/Phimodel? My expectation is that phenix.fmodel would do this with an option toggle or something. F --------------------------------------------- Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D

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No clash evaluation between symmetry mates in phenix.refine
by Keitaro Yamashita 08 Dec '10

08 Dec '10

Dear Phenix developers, I have a question about phenix.refine. I'm refining complex structure against X-ray diffraction data with packing disorder. (Some domains overlap with their symmetry mates (4-fold), so their occupancies are set to 0.25) So I want phenix.refine to ignore clashes between symmetry mates. Can phenix.refine exclude any interaction between symmetry mates from geometric term in target function: E = E(x-ray) + E(geometry)? Is there any option? Thank you very much in advance, K. Yamashita

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switch off anti-bump restrain
by Agnieszka 08 Dec '10

08 Dec '10

Hi, I have double conformation of my residue. There is a water molecule that interacts with conformation A. In the same time the water is very close to conformation B. During the refinement the water is pushed out. I try to switch off the site refinement of the water and some atoms of conformation b but it didn't work out for me. Do you know how can I switch off the anti-bump restrain of this two guys? Thank you Agnieszka Kiliszek

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ncs restraints
by Timothy Springer 08 Dec '10

08 Dec '10

Hi, I am using NCS restraints for 4 copies of a molecule at 3.05 Å. We are using the standard weights: coordinate_sigma = 0.05 b_factor_weight = 10 and the 4 molecules look identical. What are suggested values for looser restraints? One of my colleagues said that varying them did not make a difference- ncs was still very strict. Is there a way to make them less strict? Thanks, Timothy A. Springer, Ph.D.

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phaser version
by Bryan Lepore 08 Dec '10

08 Dec '10

using dev-596 from source, i see `phenix.phaser --version` is Phaser-2.2.4 (svn ) that's true for dev-601 (Intel binary) also. the phaser wiki says 2.3 is the latest, and to compile it from the phenix distribution. so... wondering how to get phaser 2.3 (before asking questions about ep_sad...) -Bryan

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anyone with a scalepack file with R3 or R32 using rhombohedral axes?
by Ralf W. Grosse-Kunstleve 08 Dec '10

08 Dec '10

I'm looking for a little help from the phenix user community. Does anyone have a scalepack output file with data in space group R3 or R32 using rhombohedral axes, i.e. unit cell parameters a=b=c, alpha=beta=gamma? What is the space group symbol in the file? I'm only interested to know this for files that are the direct output of scalepack. I'd like to verify that this part of the scalepack documentation is actually true: http://www.hkl-xray.com/hkl_web1/hkl/manual_online.pdf Page 139: 146 R3 R3 in hexagonal setting 446 H3 R3 in primitive setting 155 R32 R32 in hexagonal setting 455 H32 R32 in primitive setting If this is actually true, it is the exact opposite of what's used by the PDB. Thanks in advance for feedback. Ralf

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modelling of a novel protein
by intekhab alam 07 Dec '10

07 Dec '10

Hi I am trying to solve the structure of RNA polymerase in complex with its protein primer. The size of protein primer is 70 amino acids which i complexed with the polymerase and collected a data of the complex crysta at 2.3A. The space group is C2 and after Molecular replacemnet with full length polymerase in auto MR i got 3 subunits in ASU. The parameters of MR solution RFZ=71.5 TFZ=41.2 PAK=0 LLG=8812 suggests that the solution is correct. I used autobuild for model building which gave me the model of the polymerase as well as some extra density that i am considering as the other protein. After several round of refinemnt i have R and R free of 27and 32 respectively. My queries: 1. I want to know ,how can i improve my R as well as R free. 2. How can i model other protein as the density is weak and not complete, how can i improve the map of the other protein so that i can b ebale to trace the c-alpha backbone, i was able to model only 10 amino acids so far. Please guide. Regards -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL

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distorted (collapsed) zinc-finger motifs
by Hasan Demirci 07 Dec '10

07 Dec '10

Hi, I am in the middle of refining a 3.5 A (low resolution) ribonucleo-protein complex structure. Two of the proteins in this complex contain "zinc-finger" motif. These zinc-finger motifs instead of forming a cysteine-zinc interactions, cysteines are forming di or in these case tetra-sulfide bridges. Initially, zinc is centered but after refinement 4 cysteines come out collapsed and forming covalent bonds to each other and also distorted and zinc is not at the center anymore. Below is the phenix.refine zn_link.param file I am using. Many thanks for advice, Hasan Demirci ---------------------------------------------------------------------------- refinement.pdb_interpretation { disulfide_distance_cutoff = 0.1 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain D and resname ZN and resid 306 residue_selection_2 = chain D and resname CYS and resid 9 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain D and resname ZN and resid 306 residue_selection_2 = chain D and resname CYS and resid 12 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain D and resname ZN and resid 306 residue_selection_2 = chain D and resname CYS and resid 26 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain D and resname ZN and resid 306 residue_selection_2 = chain D and resname CYS and resid 31 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain N and resname ZN and resid 307 residue_selection_2 = chain N and resname CYS and resid 24 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain N and resname ZN and resid 307 residue_selection_2 = chain N and resname CYS and resid 27 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain N and resname ZN and resid 307 residue_selection_2 = chain N and resname CYS and resid 40 } refinement.pdb_interpretation.apply_cif_link { data_link = ZN-CYS residue_selection_1 = chain N and resname ZN and resid 307 residue_selection_2 = chain N and resname CYS and resid 43 } # data_link_ZN-CYS # loop_ _chem_link_bond.link_id _chem_link_bond.atom_1_comp_id _chem_link_bond.atom_id_1 _chem_link_bond.atom_2_comp_id _chem_link_bond.atom_id_2 _chem_link_bond.type _chem_link_bond.value_dist _chem_link_bond.value_dist_esd ZN-CYS 1 ZN 2 SG single 2.340 0.020 loop_ _chem_link_angle.link_id _chem_link_angle.atom_1_comp_id _chem_link_angle.atom_id_1 _chem_link_angle.atom_2_comp_id _chem_link_angle.atom_id_2 _chem_link_angle.atom_3_comp_id _chem_link_angle.atom_id_3 _chem_link_angle.value_angle _chem_link_angle.value_angle_esd ZN-CYS 1 ZN 2 SG 2 CB 109.000 3.000 ---------------------------------------------------------------------------------------------------- -- Hasan DeMirci, Ph.D. Postdoctoral Fellow Department of Molecular Biology, Cellular Biology & Biochemistry Brown University 185 Meeting Street Providence, RI 02912 (401) 863-3652 lab (SFH) (401) 863-6124 lab (ship st) (401) 226-7852 cell Hasan_DeMirci(a)Brown.edu demircha99(a)gmail.com

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Re: [phenixbb] Using Reference Structure in Phenix Refine
by John Pascal 05 Dec '10

05 Dec '10

Got it! Thank you, Pavel and Jeff. Best, John -- John Pascal, PhD ph 215.503.4596 Assistant Professor fx 215.923.2117 Thomas Jefferson University Biochemistry & Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ---------------------------------------------------------------- This message was sent using IMP, the Internet Messaging Program.

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Using Reference Structure in Phenix Refine
by John Pascal 04 Dec '10

04 Dec '10

Hello Everyone, I am refining against low resolution data, and I would like to use a higher resolution model that is available as a reference structure. There are multiple copies of the molecule in the asymmetric unit, each with a different chain ID. Does the reference PDB file need to contain multiple copies, such that there is a match for each chain (e.g. RESIDs and SEGIDs)? Or does phenix figure out that the reference structure refers to several copies of the molecule, and you only need a single copy in the reference molecule PDB file? Or will NCS restraints take care of this? Thanks. -John -- John Pascal, PhD ph 215.503.4596 Assistant Professor fx 215.923.2117 Thomas Jefferson University Biochemistry & Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ---------------------------------------------------------------- This message was sent using IMP, the Internet Messaging Program.

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Can PHENIX write the peak list to a PDB file?
by Joe Krahn 03 Dec '10

03 Dec '10

PHENIX has an option to find positive and negative peaks, but only lists them in the log file along with the residue they are closest to. Is it possible to write out the actual peak coordinates, such as to a PDB file? Thanks, Joe Krahn

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heavy atom search within phaser molecular replacement
by Bryan Lepore 03 Dec '10

03 Dec '10

i know that phaser will find heavy atoms in autosol, and that "MR-SAD" can be run as two separate steps, such that with some scripting, one could in principle cycle through a bunch of heavy atom searches using MR phases. however, it is not clear to me whether phaser can be run such that e.g. the refined MR solutions can have their anomalous Fourier's searched for peaks in a single MR job. seems possible in principle - before i go trying this, any comments would be appreciated.... i.e. run heavy atom searches as part of molecular replacement, for convenience. hope that makes sense. -Bryan

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Issue with phenix.superpose_maps
by Stephen Graham 03 Dec '10

03 Dec '10

Hi all, I'm trying to use phenix.superpose_maps through the GUI to get superposed maps covering the same region of NCS-related chains A and B for a figure. I have used PDB Tools to split the model into two files with only chain A or chain B. However, when I run superpose_maps specifying the two individual chains for the input PDB files but the same mtz file for the map coefficients I only get one output map (I think it overwrites the output file...). Selected input from the generated .eff file is as follows: input_files { map_1 = "/home/stepheng/blah/Native_refine_6_map_coeffs.mtz" labels_1 = 2FOFCWT,PH2FOFCWT map_2 = "/home/stepheng/blah/Native_refine_6_map_coeffs.mtz" labels_2 = 2FOFCWT,PH2FOFCWT pdb_1 = "/home/stepheng/blah/Native_refine_6_chainA.pdb" d_min_1 = None pdb_2 = "/home/stepheng/blah/Native_refine_6_chainB.pdb" d_min_2 = None output_dir = "/home/stepheng/blah" } And the end of the output is as follows: saving CCP4 map to Native_refine_6_map_coeffs_superposed.ccp4 Wrote Native_refine_6_chainA_superposed.pdb Wrote Native_refine_6_map_coeffs_superposed.ccp4 saving CCP4 map to Native_refine_6_map_coeffs_superposed.ccp4 Wrote Native_refine_6_chainB_superposed.pdb Wrote Native_refine_6_map_coeffs_superposed.ccp4 A bug? Stephen P.S. Incidentally, is there any reason you write both maps in a P1 box rather than putting map 2 on top of map 1 in the position of the first (reference) model? The latter would be more convenient as you can then re-use your carefully chosen pymol viewing matrices for all figures... -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638

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question about xtriage
by crystallogrphy 02 Dec '10

02 Dec '10

Hi, I am checking the space group of my data set using phenix.xtriage. However, the program terminated in the following scripts. Does anyone knows why and what should I do? Thanks! phenix.xtriage B13_native.sca Traceback (most recent call last): File "/usr/local/phenix-1.6.4-486/cctbx_project/mmtbx/command_line/xtriage.py", line 20, in <module> xtriage.run(args=sys.argv[1:]) File "/usr/local/phenix-1.6.4-486/cctbx_project/mmtbx/scaling/xtriage.py", line 866, in run output_file = open( params.scaling.input.parameters.reporting.log ,'w') IOError: [Errno 13] Permission denied: 'logfile.log'

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force field of PHENIX.refine ?
by chene＠umbi.umd.edu 02 Dec '10

02 Dec '10

Dear authors of PHENIX, I just want to know what force field is used by phenix.refine, especially for simulated annealing, CHARMM, AMBER, or what else? Thanks. Chen Chen Chen, PhD, Research Associate Center for Advanced Research in Biotechnology (CARB), University of Maryland Biotechnology Institute (UMBI), 9600 Gudelsky Drive, Rockville, MD 20850 240-314-6122

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distance constrain in phenix
by r n 02 Dec '10

02 Dec '10

Hi I would like to do distance restrain refinement for metal coordination. How can I do metal - protein strict distance restrain refinement in phenix? Thanks ram

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Hydrogen occupancies
by Florian Sauer 01 Dec '10

01 Dec '10

Dear all, I'm currently refining a small protein against 0.96A data using phenix.refine (1.6-486) from the GUI. Refinement without hydrogens yielded Rwork/free of 12.4/14.2. I then added H-atoms with phenix.reduce and switched to individual refinement of hydrogens which dropped the R-factors to 11.3/13.4. After this refinement the occupancies of some hydrogens dropped to 0 which makes me a little concerned. See e.g.: ATOM 752 H LYS A 45 -7.443 2.613 9.537 1.00 4.73 H ATOM 753 HA LYS A 45 -9.707 1.128 9.401 0.55 2.42 H ATOM 754 HB2 LYS A 45 -8.476 2.797 7.435 1.00 10.58 H ATOM 755 HB3 LYS A 45 -9.795 1.948 7.166 0.95 8.70 H ATOM 756 HG2 LYS A 45 -10.840 3.210 8.889 0.00 33.83 H ATOM 757 HG3 LYS A 45 -9.604 4.206 8.775 0.00 33.83 H ATOM 758 HD2 LYS A 45 -11.227 3.615 6.563 0.00 62.10 H ATOM 759 HD3 LYS A 45 -11.508 4.867 7.507 0.00 62.10 H ATOM 760 HE2 LYS A 45 -9.229 5.527 6.985 1.00 83.62 H ATOM 761 HE3 LYS A 45 -9.348 4.488 5.786 0.00 83.86 H ATOM 762 HZ1 LYS A 45 -10.909 5.758 4.853 1.00 99.63 H ATOM 763 HZ2 LYS A 45 -11.131 6.534 6.042 0.35100.44 H ATOM 764 HZ3 LYS A 45 -9.917 6.715 5.257 0.39100.08 H I would like to ask whether it is possible in phenix to group the occupancies to keep them at the same value as the rest of the residue but still refine xyz and isotropic adp of the individual hydrogens? Thank you in advance for your answers, Florian

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wander wall interaction
by subhash bihani 01 Dec '10

01 Dec '10

hi all I am working on a enzyme-ligand complex structure. For some analysis I need to remove wander wall interactions between protein and ligand. How can I do that in Phenix? Something similar to igroup statement in CNS? Thanks in adwance -- Subhash Bihani Scientific Officer Solid State Physics Division Bhabha Atomic Research Centre Trombay, Mumbai-85 Ph. 25594688 (o) alternate email: subhashbihani(a)yahoo.co.in

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phaser and threads
by Bryan Lepore 01 Dec '10

01 Dec '10

[ dev-595 ] [ GNU/Linux 2.6.31-20-generic #58-Ubuntu SMP four-processors ] [ compiled from source with --no-gui --no-apps --openmp ] i noticed phaser splits the rotation onto 2 threads - just wondering why that might be, since i used nproc=4 on the command line.... oh, hmmm... i seem to have just got this at the packing function : |python: malloc.c:4591: _int_malloc: Assertion `(unsigned long)(size) >= (unsigned long)(nb)' failed. -Bryan

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building from source : svn_checkout
by Bryan Lepore 01 Dec '10

01 Dec '10

[ dev-596 from source using --no-gui --no-apps --openmp ] README-DEV mentions ./bin/svn_checkout ... i cannot find this (but i do find bundle_update and recompile). any pointers appreciated. -Bryan

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