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Re: [phenixbb] [ccp4bb] Ligandfit - problem with ligand_start
by Terwilliger, Thomas C 25 Nov '13

25 Nov '13

Hi Danilo, The most likely problem is that the ligand_start file has to have enough atoms in it to define a fixed group in the ligand. The way that ligandfit works is that it breaks a ligand up into rigid groups connected by rotatable bonds. The ligand_start file has to contain one complete rigid group. So probably if you supply a little more of the ligand then it might run. Just to test this, can you try this test of using a ligand_start file: phenix_regression.wizards.test_resolve resolve test_fit_start And then if that gives an "OK", try this: cd test_fit_start phenix.ligandfit ligand=side.pdb ligand_start=start.pdb resolve_map.mtz quick=true Does that run? If you the look at LigandFit_run_1_/TEMP0/resolve_ligand_fit.log Does that say >ligand_start start.pdb Starting configuration of ligand will be read from: start.pdb If all that works, then try adding more atoms to your ligand_start file. Let me know if that doesn't do it! All the best, Tom T Dear all, I am working on a membrane protein covalently bound to a molecular antanna: it is known that this molecule binds to lysine residue but I do not know how many and which lysine residues it binds. 20 diffraction datasets of this protein-ligand complex have been obtained and now, I would quickly localize the ligand using the Fo-Fc map of each data set and using the information on the covalent bound protein-ligand. Ligandfit tool (PHENIX) seems to be indicated to do this; to use the information on the covalent bound, I am using the ligand_start keyword with a pdb containing a ghost atom (however present in ligand model) perfectly superposed to the lysine atom that should bind the ligand. The command used is: phenix.ligandfit data=prot.mtz model=prot.pdb ligand=lig.pdb ligand_start=lig_start.pdb input_labels="FOFCWT PHFOFCWT" \ refine_ligand=True \ nproc=32 \ cif_def_file_list=lig.cif description: - prot.mtz (data) - prot.pdb (protein without ligand) - lig.pdb (ligand containing ghost atom) - lig_start.pdb (ghost atom superpose to NZ of a lysine) - lig.cif (restrain of lig.pdb) Strangely, no ligand is found at the end of the process even reducing ligand_cc_min to 0.01. I have run the same command by using an other protein where an other ligand has been correctly fitted but, also in this case, no ligand has been detected. Conversely, without the use of ligand_start, ligandfit properly localizes the ligand. I'm doing some mistake in the use of ligand_start? Do you know an other tool to perform a ligand fitting in these conditions? Thanks for your answers. Danilo

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Ligandfit - problem with ligand_start
by Danilo Belviso 25 Nov '13

25 Nov '13

Dear all, I am working on a membrane protein bound to a antenna molecule: I know that the molecule binds to lysine residue but I do not know how many and which lysine residues it binds. 20 diffraction datasets of protein-ligand complex have been obtained and now, I would quickly localize the ligand using the Fo-Fc map of each data set and using the information on the covalent bound protein-ligand. Ligandfit tool (PHENIX) seems to be indicated to do this; to use the information on the covalent bound, I have thought to use the ligand_start keyword with a pdb containing a ghost atom (however present in ligand model) perfectly superposed to the lysine atom that should bind the ligand. The command used is: phenix.ligandfit data=prot.mtz model=prot.pdb ligand=lig.pdb ligand_start=lig_start.pdb input_labels="FOFCWT PHFOFCWT" \ refine_ligand=True \ nproc=32 \ cif_def_file_list=lig.cif description: - prot.mtz (data) - prot.pdb (protein without ligand) - lig.pdb (ligand containing ghost atom) - lig_start.pdb (ghost atom superpose to NZ of a lysine) - lig.cif (restrain of lig.pdb) At the end of the processing, ligand is not found. So, I have tryed with ligand_cc_min=0.01, but also in this case ligandfit does not give result. I have thought that no ligand is present. However, to be sure that all was correct, I have run the same command by using an other protein where an other ligand has been correctly fitted. Also in this case, no result has been obtained. Conversely, without the use of ligand_start, ligandfit properly localizes the ligand. I'm doing some mistake in the use of ligand_start? Thanks for your answers. Danilo

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using reference model with polyA chain.
by Francis Reyes 20 Nov '13

20 Nov '13

I'm refining a structure ( at low res ), so I trimmed off all the side chain atoms from my model while my reference model still has these chains. I'm receiving errors like... ----------Correcting rotamer outliers to match reference model---------- ** evaluating rotamers for working model ** A 502 ILE is missing some sidechain atoms, **skipping** A 504 LYS is missing some sidechain atoms, **skipping** A 505 VAL is missing some sidechain atoms, **skipping** I don't care about the side chain atoms, but I'd still like the reference model restraints to provide secondary structure. Does "**skipping**" mean that the reference model is no longer being considered for the particular residue? Or will I still retain my secondary structure from the reference model? Thanks, F --------------------------------------------- Francis E. Reyes PhD 215 UCB University of Colorado at Boulder

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completeness problem in phenix.refine
by Yarrow Madrona 19 Nov '13

19 Nov '13

Hi, I recently merged two data sets, one having a phase and the other Amplitudes. For some reason my completeness is always 50%. I had anomalous data but the completeness is around 100% after processing and when I do an mtz dump. I'm confused why I get a completeness of 50%. Thank you for any help you can provide. -Yarrow -- Yarrow Madrona Graduate Student Molecular Biology and Biochemistry Dept. University of California, Irvine Natural Sciences I, Rm 2403 Irvine, CA 92697

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Error: "Error running main function (INPUT): LLG completion peak selection method not recognized"
by Egor Tchesnokov 18 Nov '13

18 Nov '13

Hello, I am trying to run Phaser/SAD or Phaser/MR_SAD (GUI using defaults), but I get the following error: "Error running main function (INPUT): LLG completion peak selection method not recognized". Thank you for your help Egor

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MSE out density after phenix.refine
by Pavel Afonine 16 Nov '13

16 Nov '13

Hello, anyone experiencing problems related to MSE residues being pushed out density after phenix.refine run should update to next Phenix nightly build, which should fix this problem. Please let me know otherwise off-list. Pavel

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MR_Rosetta prerefine question
by Heather L Condurso 12 Nov '13

12 Nov '13

Dear all, I am trying to use the rosetta prerefinement fuction of MR_rosetta to complete a homology model with missing stretches of amino acids. Everything runs fine and residues are being added to fill the gaps using the fragment files, but rosetta is also altering parts of the model which should already be well placed. Is there a way to tell Rosetta only to prerefine certain residues? I've been following the command line suggestion: phenix.mr_rosetta \ seq_file=seq.dat \ data=coords1.mtz \ search_models=coords1.pdb \ run_prerefine=True \ number_of_prerefine_models=1000 \ fragment_files = test3.gz \ fragment_files = test9.gz \ rescore_mr.relax=False \ rosetta_models=100 \ ncs_copies=2 \ space_group=p212121 \ use_all_plausible_sg=False \ nproc=200 \ group_run_command=qsub Thanks, Heather Condurso condurso(a)bc.edu

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Re: [phenixbb] Phenix.Rosetta_refine on Ubuntu 12.04 LTS
by qwertgfdsa78 11 Nov '13

11 Nov '13

Hi All, I tried Jan's tutorial and finally I was able to install and ran phenix.rosetta with test set 1yjp successfully. However, when I tried my data, I always got *"terminate called after throwing an instance of 'std::bad_alloc'"* Google suggests maybe memory is not enough. I used VirutalBox and assign 8gb memory to it. My protein has ~3000 residues. I guess this will use more memory but how much RAM I need? Or the error is due to some other problem? Thank for any inputs. Lee > ------------------------------ > > Message: 3 > Date: Tue, 05 Nov 2013 14:26:00 +0100 > From: Jan Gebauer <jan.gebauer(a)uni-koeln.de> > To: PHENIX user mailing list <phenixbb(a)phenix-online.org> > Subject: Re: [phenixbb] Phenix.Rosetta_refine on Ubuntu 12.04 LTS > Message-ID: <5278F1E8.9020602(a)uni-koeln.de> > Content-Type: text/plain; charset="utf-8" > > 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 > > >

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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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