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- PDB-7o1a: Cryo-EM structure of an Escherichia coli TnaC(R23F)-ribosome comp... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7o1a | |||||||||||||||
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Title | Cryo-EM structure of an Escherichia coli TnaC(R23F)-ribosome complex stalled in response to L-tryptophan | |||||||||||||||
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![]() | TRANSLATION / ribosome / regulation / TnaC / arrest peptide / L-tryptophan / indole / stalling | |||||||||||||||
Function / homology | ![]() negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity ...negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / translational initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / DNA endonuclease activity / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / transcription antitermination / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / ribosomal large subunit assembly / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / cytoplasmic translation / rRNA binding / negative regulation of translation / ribosome / structural constituent of 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: 2.4 Å | |||||||||||||||
![]() | 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 | ![]() ![]()
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![]() | ![]() Title: 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. | |||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 3.4 MB | Display | ![]() |
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PDB format | ![]() | Display | ![]() | |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 2 MB | Display | ![]() |
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Full document | ![]() | 3.6 MB | Display | |
Data in XML | ![]() | 362.3 KB | Display | |
Data in CIF | ![]() | 588.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 12694MC ![]() 7o19C ![]() 7o1cC C: citing same article ( M: map data used to model this data |
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Similar structure data | |
EM raw data | ![]() Data 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: 26652.557 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: 2063.334 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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-RNA chain , 2 types, 2 molecules B7B8
#54: RNA chain | Mass: 2846.759 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#55: RNA chain | Mass: 24875.807 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-Non-polymers , 5 types, 2503 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/K.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/TRP.gif)
![](data/chem/img/HOH.gif)
![](data/chem/img/K.gif)
![](data/chem/img/ZN.gif)
![](data/chem/img/TRP.gif)
![](data/chem/img/HOH.gif)
#56: Chemical | ChemComp-MG / #57: Chemical | ChemComp-K / #58: Chemical | #59: Chemical | ChemComp-TRP / | #60: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | N |
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-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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Molecular weight |
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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. | ||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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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: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 59880 X / Nominal defocus max: -1600 nm / Nominal defocus min: -400 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 44 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8270 |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 358000 | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191230 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6TBV Accession code: 6TBV / Source name: PDB / Type: experimental model |