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Visiting Research Specialist Position at the University of Illinois to Study Ribosome Structure
by Yury Polikanov 27 Jul '20

27 Jul '20

The Polikanov Laboratory at the University of Illinois at Chicago (IL, USA) is looking for a talented, skillful, and highly motivated researcher to fill the *Visiting Research Specialist* position to study the structure and function of the bacterial ribosome. The research in the Polikanov Laboratory is focused on understanding the basic principles of protein synthesis in bacteria, the modes of action of ribosome-targeting antibiotics, and the mechanisms of drug resistance at a structural level. More information about Dr. Polikanov's research can be obtained from the most recent publications ( https://www.ncbi.nlm.nih.gov/sites/myncbi/yury.polikanov.1/bibliography/503…) or found on the laboratory web-page ( https://bios.uic.edu/bios/people/faculty/yury-polikanov). The successful candidate will be involved in the determination of high-resolution structures of ribosome functional complexes with various protein translation factors, tRNAs, and antibiotics using macromolecular X-ray crystallography and potentially cryo-EM techniques. *Basic qualifications:* - BS degree in chemistry, biochemistry, biophysics, or a related field; - MS degree or higher is preferred; - Minimum 3 years of laboratory research experience; - Strong background in biochemistry and molecular biology; - Practical skills with the most standard methods currently used in molecular biology, and biochemistry (such as protein expression, purification); - Experience in structural biology (cryo-EM or X-ray crystallography) is desired; - Experience in RNA biology is desired. *The successful candidate is also expected to:* - Act independently in terms of planning and executing the experiments; - Compile, analyze, and prepare data for publications; - Assist with training of graduate and undergraduate students. For fullest consideration, please submit an application and cover letter describing your scientific interests and how your previous research accomplishments and scientific interests fit with the scope of the research in the Polikanov research group at https://jobs.uic.edu/job-board/job-details?jobID=133847 by August 14, 2020. The University of Illinois at Chicago is an Equal Opportunity, Affirmative Action employer. Minorities, women, veterans, and individuals with disabilities are encouraged to apply. The University of Illinois may conduct background checks on all job candidates upon acceptance of a contingent offer. Background checks will be performed in compliance with the Fair Credit Reporting Act.

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Phaser-MR output after each component placement
by Johannes Cramer 27 Jul '20

27 Jul '20

Dear phenix board, does anyone of you know if there is an option in phaser-MR to output a pdb after each placed component? For example: I suspect there to be 5 molecules in the AU. I start a replacement with 5 molecules and phaser places 1 (I would like to inspect that), 2 (again, I would like to look at the e.g. top 10 results), 3 and so on... Is that possible? I couldn't find the option in the gui or in the phenix documentation, but maybe in the CLI? I know I can run one after the other, but that would potentially waste CPU time until I find time to inspect them. Cheers, Johannes

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Adjusting pixel size using phenix
by Noor Agip 23 Jul '20

23 Jul '20

Hi, Is there a way to adjust pixel size of a volume in phenix? I have been using ResolveCryoEM and I would now like to calibrate the pixel of the resulting map. This could be done in chimera but requires resampling the map to a RELION map of the correct pixel size. Best wishes, Noor

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Heme geometry restraint
by Yangqi Gu 21 Jul '20

21 Jul '20

Dear all, I have a question regarding the heme c geometry restraint. I am aware that heme c could undergo distortion when bound to protein, but I am not sure at what resolution I could claim such distortion (such as ruffling/bending/doming). I have a map at 3.6 A resolution and whenever I did the refinement in phenix with real space refinement, some of the hemes are distorted and I am wondering if it is justified to leave it that way or do I have to impose the flat plane geometry. If so, how should I proceed to impose such geometry? Thank you in advance! Best, Yangqi

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mr_rosetta question
by Wei Wang 18 Jul '20

18 Jul '20

Dear all, I would like to get help on a mr_rosetta run. I have a case that 5 copies of searching model was found by Phaser, and I would like to find the sixth one. In addition I would like to improve the building of the MR solution. In such case I’m considering a mr_rosetta run, with some parameters like this: phenix.mr_rosetta seq_file=seq.dat search_models=placed_5_copies.pdb already_placed=true ncs_copies=1 … My question is, should I use the already placed 5 copies model as search_models, with ncs_copies=1, or should I use the original searching model with ncs_copies=6 (in this case I guess I should get rid of the other four copies from the result of Phaser)? For seq.dat, should I use the sequence from one copy of searching model, or paste 5 copies into the seq.dat? Thanks, Wei

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ligand possibly bound to active site cysteine
by Jorge Iulek 18 Jul '20

18 Jul '20

Dear all, I am refining a structure of a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (converts glyceraldehyde 3-phosphate into D-glycerate 1,3-bisphosphate) , https://www.brenda-enzymes.org/enzyme.php?ecno=1.2.1.12 . <https://en.wikipedia.org/wiki/Glycerate_1,3-bisphosphate> It turns out that its active center cysteine presents bound ligands , covalently or not to be determined if possible (data resolution 2.51 A). I would like to get help on two issues, (1) what the ligand might be and (2) how to treat it (correct me) in phenix.refine. 1) The protein was expressed in E. coli; it had much contact with glycerol and crystallization conditions include the "ethylene-glycols-mix" ("a mixture of diethylene glycol, triethylene glycol, tetraethylene glycol, and pentaethylene glycol"). Nevertheless, no NAD cofactor was added, and there is no electron density for it. Otherwise, phosphate was also present in crystallization condition. In a previous study, I learned that glycerol might also contain minor amounts of ethylene glycol. I wonder, nevertheless, about glyceraldehyde (and note resemblance with the substrate). Catalytic mechanism includes a hemithioacetal intermediate (https://febs.onlinelibrary.wiley.com/doi/abs/10.1046/j.1432-1327.1998.25204… ) such that cysteine SD is bound covalently to a carbon. I wonder also how much this might attack an ethylene glycol and their likes. Pictures for the density are shown at for the 4 monomers of the a. u., first 4 photos: https://photos.app.goo.gl/Y7MyugqwRFD4sjgDA (blue 1 sig for e. d. maps, green 3 sig for Fourier difference maps) . Density is different among them to different degrees. The nearby threonine, in some cases, seems to interact with a blob (and it is helped by other threonine and a serine) which + - might accommodate a phosphate. I have tried to fit a number of molecules, e.g., the substrate (but not really good in all monomers for the phosphate moiety), glycerol, ethylene glycol and its di and tri (found also in other places in the structure) and now I went for glyceraldehyde (though, I have doubts that there is other - apart from the one eventually bound to S - tertiary carbon). Apart from the difficulties on searching for the best fitting molecule (and consider their intrinsic flexibility) I do not manage to establish distance between them and Cys SD (and there goes the second question). 2) I could not devise how to set a proper distance between any of the ligands and the Cysteine, be it to check for a covalent bond or to establish a van der Waals restriction. I tried: bond { action = *add delete change atom_selection_1 = chain A and resid 153 and name SG atom_selection_2 = chain N and resid 5 and name C3 symmetry_operation = None distance_ideal = 1.803 sigma = 0.1 slack = None limit = -1.0 top_out = False } Results are also show for my Glyceraldehyde trial, last 4 photos, https://photos.google.com/album/AF1QipO71L7GJYKv_MmjTc_0GzsH2xtFR_V-2ICBirPb . Note clashes. Curiously , for some of the bonds to be added, I receive the message: " Atom "HETATM 9835 O2 3GR N 5 .*. O " rejected from bonding due to valence issues." which seems to point to oxygen atoms, though I declare carbon atoms. Helps welcome, thank you. Jorge

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mr_rosetta build test
by Wei Wang 17 Jul '20

17 Jul '20

Dear all, I would like to ask a question about the testing of mr_rosetta build. Is the “phenix_regression.wizards.test_command_line_rosetta_quick" still available? I checked my early build on Mac it is there, but that build was ~3 years ago. However on a recent build on Ubuntu 18.04 the option is not available (I ran the rosetta.run_tests and it’s ok though). Regards, Wei

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occupancy_sum_constraints
by Petros Giastas 15 Jul '20

15 Jul '20

Dear all, I have a metal compound interacting with a protein and I am facing the following problem. Initially, the metal is equatorially complexed with chlorides, which are progressively exchanged by water molecules. This hydrolysis is clear from our spectroscopic analysis, but also from the anomalous maps, which show weaker peaks for the most exposed chlorides. The question regarding phenix.refine is: How can I refine the occupancies of the alternative species (chlorides and water molecules) and to constrain their sum into the occupancy of the metal, which is also under refinement? I cannot find the proper selection syntax to deal with this situation. Can you help me on that? Thank you in advance. Petros P.S. I remember I could treat this kind of problem with shelxl but it has been a lot of time since I last used shelxl for high resolution protein refinement and I would not like to go back.

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PhD position (Denmark), Structural biology with a focus on sterol uptake mechanisms
by Bjørn Panyella Pedersen 13 Jul '20

13 Jul '20

Dear colleagues, We have a open PhD position my my group. Please pass along to anyone who might be interested. Thanks! Deadline is July 31st. /Bjørn STRUCTURAL AND FUNCTIONAL STUDIES of eukaryotic membrane proteins involved in sterol transport (http://pedersenlab.dk/source/phd_position_2020.pdf) A PhD position is available in membrane protein biochemistry and structural biology in the research group of Dr. Bjorn P Pedersen at the Department of Molecular Biology and Genetics, Aarhus University, Denmark. The group studies the structure-function relationship of membrane proteins involved in metabolite uptake, primarily sterol and sugar uptake, as well as their regulation from a structural perspective. The PhD project will involve expression, purification and structural/functional characterization of membrane proteins involved in sterol transport and homeostasis (Winkler et al. Cell 179 (2019)). Methods learned and used will be cryo-electron microscopy and X-ray crystallography, as well as various biochemical methods to elucidate function. The project is fully funded by the Danish Council for Independent Research, and will be based in the laboratory of Dr. Bjorn P. Pedersen (http://pedersenlab.dk). It would ideally suit an outstanding biochemistry graduate with a strong interest in structural biology. The candidate should have molecular biology, protein production and purification experience. Experience with membrane proteins or protein structure determination will be an advantage. Full training will be provided in all necessary areas of protein biochemistry and structural biology. Good English language skills are essential. Salary will be according to the collective agreement between the Danish Confederation of Professional Associations and the Danish State. The position will be filled as soon as a suitable candidate is identified. Deadline is July 31st. Applicant should send a CV, cover letter and names of two referees to Dr. Bjorn P Pedersen (bpp(a)mbg.au.dk).

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phenix writing files with wrong job number
by Green, Todd Jason 09 Jul '20

09 Jul '20

Hello All- Phenix has started to write files from each job with incorrect job number notation. It puts them into the correct directory (ie. with correct job number, ex. Refine_23) but with file names having wrong job number (ie. where they are job number - 1, ex. protein_refine_22.pdb). This causes issues with the gui. I can edit the names post run, but is there a permanent fix. Just start a new project? Probably upgrade to the new version is the first choice. FYI I'm running 1.17.1-3660-000 on a macbook. Seems to have just started spontaneously. Thanks for the help- Todd

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