- phenixbb - phenix-online.org

Phenix.Refine fix WXC has no effect
by White, Mark 17 Aug '20

17 Aug '20

Hello, I am refining a 3.5 Å structure using the latest stable version of Phenix.Refine. After switching to the latest version the Bond RMSDs exploded to ~ 0.035 Å, when using optimize wxc. Turning off Optimize and using the "Fix WXC" parameters in the GUI with increasingly smaller values had almost no effect on the final bond rmsds, which seems to explode from reasonable values in the "3_xyz" step of refinement. Am I missing something? Best regards, Mark Mark Andrew White, Ph.D. Associate Professor of Biochemistry & Molecular Biology, Manager, SCSB Macromolecular X-ray Laboratory UTMB, Galveston, TX http://xray.utmb.edu

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Phenix.refine on coordinate-optimised structures
by Joey Farrell 07 Aug '20

07 Aug '20

Hello, I am trying to design a set of input parameters that I will be passing through the phenix.refine function. The structures that I am working with have already had their X,Y, and Z orthogonal Å coordinates optimised via separate protocol. I am looking for advice on which strategies and parameter values to use with phenix.refine to improve the crystallographic R-factors of such structures while preserving their earlier optimisation in real space. I have been having some success using “strategy = tls+individual_adp+occupancies” (where tls = “chain A”, tls = “chain B”, etc.) as well as adjusting the wxu_scale value until R-work and R-free are minimised. Are there other strategies or phenix.refine functionalities that may also prove helpful with this end in mind? I believe there may be ways to further improve R-work and R-free with this software that I am not yet aware of. Thank you for your help! Best, Joey

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Phenix Real_Space_Refine and Torsion NCS restraints
by Mark A. White 04 Aug '20

04 Aug '20

Hello, Are there plans to include Torsion Angle NCS restraints in the Phenix.Real_Space_Refine module? At low resolution (~ 3-4Å) I would like to include NCS restraints, but my molecule is flexible and the strict NCS constraints used in the Real_Space_Refine module are not appropriate. I am currently using phenix-1.18.2-3874. Best regards, Mark

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map to model - DNA model building
by Manoj saxena 04 Aug '20

04 Aug '20

Hello, I am trying to fit a cryo-EM map of a DNA protein complex (3.3 A) using the map to model in Phenix 1.18.2.3874, with a DNA sequence input in fasta format and the map as a .ccp4 file. However the output model fitted in the map I am getting is many fragmented PDB files and only one chain of DNA. What is the best way to get a higher fraction of the DNA model fitted into the map, can I switch off the segmentation of map and get both strands fitted into the map. Or the only option is to manually build? Thanks for your help Regards Manoj Saxena

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Model moving out of map
by Shanti Pal Gangwar 03 Aug '20

03 Aug '20

Dear all, I am trying to refine a Cryo-EM map at a decent resolution. The model was fit into the map in chimera and refined against the same map in Real space refinement in Phenix but after the run, a significant part of the model moved out of the density. I would appreciate any suggestion/strategy to solve this issue. Thank you Shanti Pal

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Postdoc position at UT Health Science Center, San Antonio, Texas
by Yogesh Gupta 02 Aug '20

02 Aug '20

The laboratory of Dr. Yogesh Gupta at University of Texas Health Science Center in San Antonio is seeking an enthusiastic Postdoctoral Fellow to study the structure and function of nucleoproteins in infectious diseases and cancer. We combine structural biology tools (X-ray, cryo-EM, NMR) with cell-based methods to address mechanistic questions and exploit this knowledge for drug development. Please visit below websites to learn about our research program – https://www.nature.com/articles/s41467-020-17496-8 *http://ccri.uthscsa.edu/YGupta.html <http://ccri.uthscsa.edu/YGupta.html>* Preference will be given to applicants with demonstrated experience in macromolecular crystallography. Candidates are expected to possess strong organizational, interpersonal skills, and ability to work as part of a team. An ideal candidate should hold (or soon expect to hold) a Ph.D. or equivalent degree in biochemistry/biophysics/structural biology and have no more than 2 years of postdoc experience. Salary is competitive. San Antonio offers affordable cost of living and there is no state income tax in Texas. If interested in applying, please send a short description of your research accomplishments, and CV with a list of 3 references to Dr. Yogesh Gupta (email: *guptay(a)uthscsa.edu <guptay(a)uthscsa.edu>*). The Greehey Children’s Cancer Research Institute is a unique specialized cancer research center focusing on basic and translational research in childhood cancer and occupies a state-of-the-art 100,000 sq. foot research facility on the university’s Greehey Academic and Research Campus. San Antonio is the nation’s seventh largest city and is located at the edge of the beautiful Texas Hill Country. San Antonio offers a rich, multi-cultural community, affordable cost of living, excellent weather and a thriving biomedical industry.* All Postdoctoral appointments are designated as security sensitive positions. The University of Texas Health Science Center at San Antonio is an Equal Opportunity/Affirmative Action Employer including protected veterans and persons with disabilities.* Thank you very much for your time. Yogesh -- Yogesh Gupta, PhD Assistant Professor of Biochemistry & Structural Biology PI, Greehey Children's Cancer Research Institute University of Texas Health Science Center 8403 Floyd Curl Drive, San Antonio, Texas, USA 78229 http://ccri.uthscsa.edu/YGupta.html

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Fwd: Sanofi Postdoctoral Fellowship: Computational Scientist for Antibody Engineering (Greater Boston, MA)
by yanfeng zhou 31 Jul '20

31 Jul '20

If you have strong interest in one of the following areas, please continue to read and check the link for more details. 1. Developing new algorithms to revolutionize biologics discovery 2. Challenging yourself with large and unique datasets and converting your learnings to tools/products that can help patients 3. Engineering of antibody, nanobody, and other biotherapeutics modalities 4. Learning how scientists from various training backgrounds collaborate together to discover and deliver faster and better biologics treatments. The Protein Engineering group within Sanofi Large Molecule Research Department is seeking a highly talented Computational Scientist for a postdoctoral position. We use leading-edge structural biology and computational tools to support biologics project needs from Sanofi’s multi-targeting, multi-modality research pipeline. Our team has a great track record of patents and publications, including Science, Nature Cancer, and Nature Communications, in the area of biologics characterization, antibody engineering and multi-specific antibodies. This postdoc project will focus on developing a machine learning approach to predict epitope-paratope, given any antibody-antigen pair, by combining in-depth understanding of structure biology, antibody engineering, and large datasets both internally and externally. The successful candidate will be with Ph. D. degree in related area and has an advanced understanding and practical experience with developing and applying complex machine learning solutions for structural biology data. The successful candidate will gain deeper insight into drug development process and transform AI/ML algorithms into accelerating biologics discovery and development process. Application Link: https://sanofi.wd3.myworkdayjobs.com/en-US/StudentPrograms/job/Framingham-M… *About Sanofi:* Every day, Sanofi's 100,000 employees are committed to improve the lives of people around the world, with sustainable and responsible solutions and initiatives. Sanofi is recognized as one of the leading biopharmaceutical companies in the world and is pioneering innovation in biologics with the first approved nanobody drug, the first clinical stage tri-specific antibody, and a novel genetic codon expansion platform for new engineering capabilities. Details of the organization and the company’s mission and goals can be found on our website (http://www.sanofi.us/l/us/en/index.jsp). ------------------------------ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 -- Yanfeng Zhou, Ph.D. Email: YanfengZhou.pku(a)gmail.com <yanfengzhou.pku(a)gmail.com>

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postdoctoral position at the Institute for Protein Innovation in Boston, USA
by Christopher Bahl 29 Jul '20

29 Jul '20

The Bahl Lab <http://function-structure.org/> at the Institute for Protein Innovation (IPI) in Boston is recruiting Postdoctoral Fellows. We use protein design to accelerate drug discovery and to engineer new lead therapeutic molecules. In addition to traditional academic collaborations, we work closely with pharmaceutical and biotech companies, as well as venture capital firms. We are looking for translationally-minded scientists who want their work to have a direct impact on the world. We will provide training in computational protein design using Rosetta, including de novo protein design and the design of protein function and biophysical properties. Postdoctoral Fellows at IPI leverage our high-throughput automated laboratory infrastructure. We are especially excited to expand our work in the following research areas: - prediction and design of protein developability for therapeutic applications - engineering of de novo miniprotein/peptide lead therapeutics for oncology, infectious disease, hypertension and diabetes, etc. - antibody design, including de novo paratope design - enzyme design Strong candidates will have demonstrated expertise in one or more of the following: - protein biochemistry: protein production and biophysical characterization, enzymology - structural biology: cryo-EM, X-ray crystallography, NMR - immuno-oncology: cell culture models, fluorescence activated cell sorting - traditional protein engineering: directed evolution, library construction, yeast and/or phage display - computational biology: machine learning, data science, molecular dynamics and/or modeling If you are excited to join a team of protein designers working to tackle audacious projects aimed directly at improving human health, please send your CV to: chris.bahl(a)proteininnovation.org About IPI The Institute for Protein Innovation is a non-profit organization that is advancing protein science to accelerate research and improve human health. The Institute is located at Harvard Medical School. IPI uniquely combines the freedom of academic exploration with the high-throughput scaling of industry to take on transformative protein-based initiatives that no other laboratory or organization can or will pursue. IPI is deploying state-of-the-art technologies to build a repository of powerful protein tools and reagents and share them through licensing and collaboration. These will enable new biomedical and therapeutic discovery for researchers in academia and industry alike. This constellation of vanguard technology and expertise positions IPI to train the next generation of entrepreneurs and protein scientists to lead the discovery of new medicines. The IPI is an equal opportunity employer and prohibits discrimination based on ethnicity, religion, sexual orientation, gender identity/expression, national origin/ancestry, age, disability, socioeconomic status, marital or veteran status, etc. Candidates from underrepresented backgrounds are especially encouraged to apply. -- PLEASE NOTE: This message, including any attachments, may include privileged, confidential and/or inside information belonging to IPI. Any distribution or use of this communication by anyone other than the intended recipient(s) is strictly prohibited and may be unlawful. If you are not the intended recipient, please notify the sender by replying to this message and then delete it from your system. Thank you.

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Re: [phenixbb] Phaser-MR output after each component placement
by Randy Read 28 Jul '20

28 Jul '20

Hi Boaz, At the moment, that is indeed the only thing you can do with the partial solution information from the log file. In the CCP4 environment, you can cut and paste lines from the log file to make a .sol file, and use that to run something like a refinement job to produce a PDB and MTZ for a partial solution, but the Phenix environment uses Python pickle files to store the transformations, so that option doesn’t apply. (Unless you run Phaser from a script, in which case the Phenix and CCP4 versions work the same.) At one point, Phaser was writing out a PDB file for the top solution at every point in the process, but we felt that led to clutter and that it was confusing. There’s a major rewrite going on at the moment, and the new phasertng will primarily be run through the phaser.voyager pipeline. One of the design criteria here was to be able to examine branches that weren’t taken or intermediate points in the ongoing search, and to be able to work with them while the overall job is still running. So there will be a better option in the hopefully not too distant future! Best wishes, Randy ----- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: +44 1223 336500 The Keith Peters Building Fax: +44 1223 336827 Hills Road E-mail: rjr27(a)cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > On 27 Jul 2020, at 23:02, Boaz Shaanan <bshaanan(a)bgu.ac.il> wrote: > > Hi, > The closest option that comes to mind to do what you're looking for is to take the Euler angles+translations for each of the monomers in the .sol file (or at the top of the final pdb file) and apply each of them to the initial model using any transformation program of your choice. > I am more familiar with running Phaser using the ccp4 gui but it shouldn't be very different under Phenix. > Phaser people may well suggest a more elegant option. > Stay safe, > Boaz > > Boaz Shaanan, Ph.D. > Department of Life Sciences > Ben Gurion University of the Negev > Beer Sheva > Israel > > On Jul 27, 2020 16:02, Johannes Cramer <johannes.cramer(a)gmail.com> wrote: > 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 > _______________________________________________ > phenixbb mailing list > phenixbb(a)phenix-online.org > http://phenix-online.org/mailman/listinfo/phenixbb > Unsubscribe: phenixbb-leave(a)phenix-online.org

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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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The 5'end guanosine triphosphate is not linked to the rest of RNA model by Phenix refine
by Zixian Li 08 Jul '20

08 Jul '20

Dear Phenix Community, I'm at beginner level of using Phenix. I have difficulty to refine a protein-RNA complex in Phenix. I refined the protein model without RNA first, then I built the RNA model into the difference density map in Coot through Calculate>Other Modelling Tools>Ideal DNA/RNA, where I input the sequence and fit nucleotides into the density. At the 5' end it's guanosine triphosphate (GTP) but the modeler tool in Coot only generates nucleoside monophosphate. So I built the RNA model from the second nucleotide at 5' end downstream until where RNA density was too poor to model. I did the real space refine zone in Coot to fit this main part of the RNA into its density the best possibly. For the GTP at 5' end, I realized there is GTP.cif restraint file in Phenix monomer library. So I obtained an arbitrary pdb file for GTP using eLBOW and corrected atom names matching the definition in GTP.cif file. Then I can also fit GTP into the RNA density at 5' end using real space refine zone in Coot. Now I have a broken RNA model with GTP unlinked to the ribose-phosphate backbone. For Phenix refinement, I supplied a cif_link_GTP-G.params file to define the linkage for the two RNA chains in the complex: refinement.pdb_interpretation { apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } } After refinement, I noticed O3' of GTP appears connected to P of the following nucleotide G in the refined model in Coot. However, if I do the real space refine zone in Coot, the linkage becomes broken, i.e., I cannot refine the RNA model as a whole: Coot only allows separate real space refine for GTP and the rest. It seems Coot still cannot recognize GTP and the rest as one body. In the refinement settings there is a button for Automatic linking options. Interestingly, I noticed if I leave the automatic covalent linking as default, Phenix writes Link records in the output pdb after refinement. But it also links one of the protein residues to the GTP in chain D. In order to avoid the unwanted intermolecular linkage, I clicked Link none for Automatic linking options, then there is no any link records written in the output pdb despite of the defined linkage in cif_link_GTP-G.params file. So it seems for me Phenix ignores the input cif_link_GTP-G.params file, it writes Link records only if the Automatic linking option is activated. I worried whether it could be due to the syntax of the cif_link_GTP-G.params file. So I tried the following syntax in two separate files, the same issue was observed. cif_link_GTP-G_chC.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain C and resname GTP and resid 1 residue_selection_2 = chain C and resname G and resid 2 } cif_link_GTP-G_chD.params refinement.pdb_interpretation.apply_cif_link { data_link = p residue_selection_1 = chain D and resname GTP and resid 1 residue_selection_2 = chain D and resname G and resid 2 } I also need to point out that p linkage is pre-defined in the list of links (mon_lib_list.cif) in Phenix monomer library. I apologize for this long explanation of the problem. I hope it is clear. I really appreciate your advice in advance. Sincerely, Zixian Li

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Heteroatoms addition to crystal structures
by Shirin Akbar 06 Jul '20

06 Jul '20

Dear all, I am using the phenix.real_space_refine program for refinement of crystal structure in cryo-EM map (ribosome map, 3.4 A resolution). Prior to real_space_refinement I had removed all the heteroatoms. What is the correct way to add the cations (eg. Mg2+ ions) to the structure and how do I do it? Can anyone kindly suggest.. Thanks & Regards Shirin -- *Shirin Akbar*

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real-space refinement resolution
by Alexei Gorelik 29 Jun '20

29 Jun '20

Hi, When refining a model against a density-modified cryo-EM map, should one use the resolution of the original or modified map? Thanks

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Refining cytochrome structure with hemes
by Yangqi Gu 29 Jun '20

29 Jun '20

Dear Phenix developers. I have a simple question regarding the real space refinement. I am working on a cytochrome structure and I would like to know how to refine the model with hemes present against the map? Also, how do I create vinyl bonds at my cysteine positions to connect the hemes? More ideally, could I refine the heme geometry as well? Thank you in advance! Best, Yangqi

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CryoEM Post-Doctoral Position
by Hasan, Syed Saif 25 Jun '20

25 Jun '20

Interested in exploring the world of molecular espionage? Want to find out how viruses hitchhike between organelles during assembly? Join my lab as a post-doctoral researcher to decipher the secret life of epidemic causing viruses at the atomic scale. We use high-resolution single particle cryoEM and X-ray crystallography to investigate how viruses such as Dengue, SARS-CoV-2, and pox viruses take over host membranes during intra-cellular progeny assembly. We are located in Rockville MD in the Institute for Bioscience and Biotechnology Research (IBBR; https://www.ibbr.umd.edu/profiles/s-saif-hasan), which is 20 miles north-west of Washington DC. In IBBR, you will have in-house access to a 200 keV Talos Arctica with a Falcon 3EC DED and a 200 keV Glacios with a K3 DED and a Volta Phase Plate (VPP). We have off-site access to a Titan Krios microscope equipped with DED and VPP. For X-ray crystallography, we have access to a Mosquito crystallization robot. Data collection is usually performed at synchrotrons. Our research is highly collaborative and is supported by wonderful colleagues who help us out with their expertise in NMR spectroscopy, lipidomics, HDX-MS, MD simulations, computer assisted drug design, and cellular imaging. If you are interested, send me your CV. Lets talk. ************************************************************************************************ The University of Maryland, Baltimore is an Equal Opportunity, Affirmative Action employer. Minorities, women, individuals with disabilities and protected veterans are encouraged to apply. ************************************************************************************************ Saif S. Saif Hasan, PhD Assistant Professor Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine 108 N. Greene Street, Baltimore MD 21201 https://www.medschool.umaryland.edu/profiles/Hasan-S-Saif-Hasan-Syed-Saif/ Associate Member Program in Oncology, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center (UMGCCC) 22 S. Greene Street, Baltimore MD 21201 Office and Laboratory Address: Center for Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research (IBBR) 9600 Gudelsky Drive, Rockville MD 20850 https://www.ibbr.umd.edu/profiles/s-saif-hasan Phone: 240-314-6396 Fax: 240-314-6225

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Energy minimization and explicit solvent model
by Andre LB Ambrosio 24 Jun '20

24 Jun '20

Dear colleagues, could you please suggest a computational tool to perform energy minimization of a crystal structure under explicit solvent conditions (i.e, types and concentrations of ions, pH)? I want to observe the solvent effects on the conformational space around a starting model, all-atom, while preserving its geometry (I guess it can be better expressed as relaxation). Thank you in advance. -- Andre LB Ambrosio

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