November 2013 - phenixbb - phenix-online.org

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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