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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 05 Nov '13

05 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 05 Nov '13

05 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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