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Yorodumi- PDB-7o19: Cryo-EM structure of an Escherichia coli TnaC-ribosome complex st... -
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Open data
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Basic information
| Entry | Database: PDB / ID: 7o19 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Title | Cryo-EM structure of an Escherichia coli TnaC-ribosome complex stalled in response to L-tryptophan | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Keywords | TRANSLATION / ribosome / regulation / TnaC / arrest peptide / L-tryptophan / indole / stalling | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Function / homology | Function and homology informationtranscriptional attenuation by ribosome / negative regulation of cytoplasmic translational initiation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / positive regulation of ribosome biogenesis ...transcriptional attenuation by ribosome / negative regulation of cytoplasmic translational initiation / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / positive regulation of ribosome biogenesis / RNA-binding transcription regulator activity / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / regulation of mRNA stability / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / ribosome assembly / regulation of DNA-templated transcription elongation / transcription elongation factor complex / cytosolic ribosome assembly / response to reactive oxygen species / DNA endonuclease activity / transcription antitermination / translational initiation / regulation of cell growth / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosome biogenesis / large ribosomal subunit / regulation of translation / ribosome binding / transferase activity / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / small ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / structural constituent of ribosome / ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Biological species | ![]() | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Authors | van der Stel, A.X. / Gordon, E.R. / Sengupta, A. / Martinez, A.K. / Klepacki, D. / Perry, T.N. / Herrero del Valle, A. / Vazquez-Laslop, N. / Sachs, M.S. / Cruz-Vera, L.R. / Innis, C.A. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Funding support | France, United States, 4items
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Citation | Journal: Nat Commun / Year: 2021Title: Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling. Authors: Anne-Xander van der Stel / Emily R Gordon / Arnab Sengupta / Allyson K Martínez / Dorota Klepacki / Thomas N Perry / Alba Herrero Del Valle / Nora Vázquez-Laslop / Matthew S Sachs / Luis R ...Authors: Anne-Xander van der Stel / Emily R Gordon / Arnab Sengupta / Allyson K Martínez / Dorota Klepacki / Thomas N Perry / Alba Herrero Del Valle / Nora Vázquez-Laslop / Matthew S Sachs / Luis R Cruz-Vera / C Axel Innis / ![]() Abstract: Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive ...Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Structure visualization
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| Structure viewer | Molecule: Molmil Jmol/JSmol |
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Downloads & links
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Download
| PDBx/mmCIF format | 7o19.cif.gz | 3.4 MB | Display | PDBx/mmCIF format |
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| PDB format | pdb7o19.ent.gz | Display | PDB format | |
| PDBx/mmJSON format | 7o19.json.gz | Tree view | PDBx/mmJSON format | |
| Others | Other downloads |
-Validation report
| Summary document | 7o19_validation.pdf.gz | 794.6 KB | Display | wwPDB validaton report |
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| Full document | 7o19_full_validation.pdf.gz | 2.4 MB | Display | |
| Data in XML | 7o19_validation.xml.gz | 302.7 KB | Display | |
| Data in CIF | 7o19_validation.cif.gz | 478.9 KB | Display | |
| Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/o1/7o19 ftp://data.pdbj.org/pub/pdb/validation_reports/o1/7o19 | HTTPS FTP |
-Related structure data
| Related structure data | ![]() 12693MC ![]() 7o1aC ![]() 7o1cC C: citing same article ( M: map data used to model this data |
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| Similar structure data | |
| EM raw data | EMPIAR-10695 (Title: Cryo-EM structure of an Escherichia coli TnaC-ribosome complex stalled in response to L-tryptophanData size: 6.9 TB Data #1: Raw multiframe micrographs of TnaC-WT-70S ribosome stalled on L-Trp [micrographs - multiframe] Data #2: Raw multiframe micrographs of TnaC-R23F-70S ribosome stalled on L-Trp (dataset B) [micrographs - multiframe] Data #3: Raw multiframe micrographs of TnaC-R23F-70S ribosome stalled on L-Trp (dataset A) [micrographs - multiframe]) |
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Links
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Assembly
| Deposited unit | ![]()
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Components
-Ribosomal RNA ... , 3 types, 3 molecules AABABB
| #1: RNA chain | Mass: 497404.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
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| #22: RNA chain | Mass: 939607.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #23: RNA chain | Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
-30S ribosomal protein ... , 20 types, 20 molecules ABACADAEAFAGAHAIAJAKALAMANAOAPAQARASATAU
| #2: Protein | Mass: 26781.670 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
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| #3: Protein | Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #4: Protein | Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #5: Protein | Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #6: Protein | Mass: 15727.512 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #7: Protein | Mass: 20055.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #8: Protein | Mass: 14146.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #9: Protein | Mass: 14886.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #10: Protein | Mass: 11755.597 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #11: Protein | Mass: 13870.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #12: Protein | Mass: 13814.249 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #13: Protein | Mass: 13128.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #14: Protein | Mass: 11677.637 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #15: Protein | Mass: 10290.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #16: Protein | Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #17: Protein | Mass: 9724.491 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #18: Protein | Mass: 9005.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #19: Protein | Mass: 10455.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #20: Protein | Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
| #21: Protein | Mass: 8524.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
+50S ribosomal protein ... , 29 types, 29 molecules BCBDBEBFBGBHBIBJBKBLBMBNBOBPBQBRBSBTBUBVBWBXBYBZB0B1B2B3B4
-Protein/peptide , 1 types, 1 molecules B5
| #53: Protein/peptide | Mass: 2073.354 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() Plasmid details: Synthesized by in-vitro translation reaction References: UniProt: A0A6D2WEB5 |
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-RNA chain , 2 types, 2 molecules B7B8
| #54: RNA chain | Mass: 2172.347 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() |
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| #55: RNA chain | Mass: 24846.746 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
-Non-polymers , 4 types, 967 molecules 






| #56: Chemical | ChemComp-MG / #57: Chemical | #58: Chemical | ChemComp-TRP / | #59: Water | ChemComp-HOH / | |
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-Details
| Has ligand of interest | N |
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| Has protein modification | Y |
-Experimental details
-Experiment
| Experiment | Method: ELECTRON MICROSCOPY |
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| EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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| Source (natural) |
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| Source (recombinant) | Organism: synthetic construct (others) | ||||||||||||||||||||||||||||||||||||||||||
| Buffer solution | pH: 7.5 Details: All buffers used during the preparation contained 2 mM L-tryptophan. | ||||||||||||||||||||||||||||||||||||||||||
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| Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Sample was purified using sucrose gradient ultracentrifugation, diluted to 200 nM and applied to grids. | ||||||||||||||||||||||||||||||||||||||||||
| Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||||||||||||||||||||||||
| Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
| Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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| Microscopy | Model: FEI TALOS ARCTICA |
| Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
| Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 45000 X / Calibrated magnification: 55127 X / Nominal defocus max: -2000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm |
| Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
| Image recording | Electron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9170 |
| Image scans | Movie frames/image: 38 / Used frames/image: 1-38 |
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Processing
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| CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
| Particle selection | Num. of particles selected: 905648 | ||||||||||||||||||||||||||||||||||||||||||||
| Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
| 3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93588 / Num. of class averages: 2 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
| Atomic model building | Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||||||||||||||||||||||
| Atomic model building | PDB-ID: 6TBV Accession code: 6TBV / Source name: PDB / Type: experimental model |
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