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Yorodumi- EMDB-5386: tmRNA translocation and MLD-loading on the ribosome: a 70S-tmRNA-... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5386 | |||||||||
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Title | tmRNA translocation and MLD-loading on the ribosome: a 70S-tmRNA-EF-G complex | |||||||||
Map data | Overall view of the 70S-tmRNA-SmpB-EF-G complex | |||||||||
Sample |
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Keywords | trans-translation / MLD-loading / translocation / tmRNA / EF-G / SmpB | |||||||||
Function / homology | Function and homology information stringent response / 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 ...stringent response / 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 / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / large ribosomal subunit / small ribosomal subunit rRNA binding / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / ribosomal large subunit assembly / small ribosomal subunit / transferase activity / large ribosomal subunit rRNA binding / 5S rRNA binding / cytosolic small ribosomal subunit / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / 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 / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 8.3 Å | |||||||||
Authors | Ramrath DJF / Yamamoto H / Rother K / Wittek D / Pech M / Mielke T / Loerke J / Scheerer P / Ivanov P / Teraoka Y ...Ramrath DJF / Yamamoto H / Rother K / Wittek D / Pech M / Mielke T / Loerke J / Scheerer P / Ivanov P / Teraoka Y / Shpanchenko O / Nierhaus KH / Spahn CMT | |||||||||
Citation | Journal: Nature / Year: 2012 Title: The complex of tmRNA-SmpB and EF-G on translocating ribosomes. Authors: David J F Ramrath / Hiroshi Yamamoto / Kristian Rother / Daniela Wittek / Markus Pech / Thorsten Mielke / Justus Loerke / Patrick Scheerer / Pavel Ivanov / Yoshika Teraoka / Olga Shpanchenko ...Authors: David J F Ramrath / Hiroshi Yamamoto / Kristian Rother / Daniela Wittek / Markus Pech / Thorsten Mielke / Justus Loerke / Patrick Scheerer / Pavel Ivanov / Yoshika Teraoka / Olga Shpanchenko / Knud H Nierhaus / Christian M T Spahn / Abstract: Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the ...Bacterial ribosomes stalled at the 3' end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD-SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome-tmRNA complexes at low to intermediate resolution, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S-tmRNA-SmpB-EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TI(POST)) presents the TLD-SmpB module in an intrasubunit ap/P hybrid site and a tRNA(fMet) in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA-SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5386.map.gz | 80.2 MB | EMDB map data format | |
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Header (meta data) | emd-5386-v30.xml emd-5386.xml | 11.6 KB 11.6 KB | Display Display | EMDB header |
Images | emd_5386_1.png | 634.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5386 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5386 | HTTPS FTP |
-Validation report
Summary document | emd_5386_validation.pdf.gz | 327.5 KB | Display | EMDB validaton report |
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Full document | emd_5386_full_validation.pdf.gz | 327.1 KB | Display | |
Data in XML | emd_5386_validation.xml.gz | 6.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5386 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5386 | HTTPS FTP |
-Related structure data
Related structure data | 4v6tMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5386.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Overall view of the 70S-tmRNA-SmpB-EF-G complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.26 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of 70S-tmRNA-SmpB-EF-G
Entire | Name: Complex of 70S-tmRNA-SmpB-EF-G |
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Components |
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-Supramolecule #1000: Complex of 70S-tmRNA-SmpB-EF-G
Supramolecule | Name: Complex of 70S-tmRNA-SmpB-EF-G / type: sample / ID: 1000 / Details: the sample was flash frozen in liquid ethane / Oligomeric state: Monomer / Number unique components: 5 |
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Molecular weight | Experimental: 3 MDa / Theoretical: 3 MDa / Method: Sedimentation |
-Supramolecule #1: Ribosome
Supramolecule | Name: Ribosome / type: complex / ID: 1 / Name.synonym: 70S-tmRNA-EFG / Details: the complex was stalled with fusidic acid / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: CAN20 |
Molecular weight | Experimental: 3 MDa / Theoretical: 3.05 MDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.15 mg/mL |
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Buffer | pH: 7.6 Details: 20 mM HEPES-KOH, pH 7.6, 4.5 mM Mg(Ac)2, 150 mM NH4Ac, 4 mM b-mercaptoethanol, 2 mM spermidine, and 0.05 mM spermine |
Staining | Type: NEGATIVE / Details: flash frozen in liquid ethane |
Grid | Details: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 96.15 K / Instrument: OTHER / Details: Vitrification instrument: FEI VITROBOT / Method: blot for 10 seconds before plunging |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Temperature | Min: 77.15 K / Max: 81.15 K / Average: 77.15 K |
Date | Dec 8, 2009 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.7 µm / Number real images: 302 / Average electron dose: 20 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 39000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000 |
Sample stage | Specimen holder: Cartridge / Specimen holder model: GATAN HELIUM |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Defocus groups |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 68842 |
-Atomic model buiding 1
Initial model | PDB ID: 3r8s |
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Software | Name: Chimera |
Details | Protocol: Rigid Body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation |
Output model | PDB-4v6t: |