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Yorodumi- EMDB-5775: Cryo-EM reconstruction of a authentic 70S-tRNA2-mRNA-EF-G translo... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5775 | |||||||||
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Title | Cryo-EM reconstruction of a authentic 70S-tRNA2-mRNA-EF-G translocation intermediate | |||||||||
Map data | reconstruction of an in vitro prepared 70S-tRNA2-mRNA-EF-G complex | |||||||||
Sample |
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Keywords | ribosome / translocation / tRNAs / mRNA / EF-G / head-swivel | |||||||||
Function / homology | Function and homology information ribosome disassembly / guanosine tetraphosphate binding / translational elongation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding ...ribosome disassembly / guanosine tetraphosphate binding / translational elongation / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, 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 / translational termination / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / translation elongation factor activity / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / transcription elongation factor complex / positive regulation of RNA splicing / DNA endonuclease activity / : / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / regulation of cell growth / maintenance of translational fidelity / DNA-templated transcription termination / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / ribosome biogenesis / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / negative regulation of translation / tRNA binding / molecular adaptor activity / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / GTPase activity / negative regulation of DNA-templated transcription / GTP binding / protein homodimerization activity / 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 / Resolution: 6.8 Å | |||||||||
Authors | Ramrath DJF / Lancaster L / Sprink T / Mielke T / Loerke J / Noller HF / Spahn CMT | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2013 Title: Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation. Authors: David J F Ramrath / Laura Lancaster / Thiemo Sprink / Thorsten Mielke / Justus Loerke / Harry F Noller / Christian M T Spahn / Abstract: During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is ...During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA. | |||||||||
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_5775.map.gz | 89.6 MB | EMDB map data format | |
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Header (meta data) | emd-5775-v30.xml emd-5775.xml | 12.6 KB 12.6 KB | Display Display | EMDB header |
Images | emd_5775.png | 107.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5775 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5775 | HTTPS FTP |
-Related structure data
Related structure data | 4v7bMC 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_5775.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | reconstruction of an in vitro prepared 70S-tRNA2-mRNA-EF-G complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 : ribosomal 70S-EF-G complex at an authentic intermediate state dur...
Entire | Name: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation |
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Components |
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-Supramolecule #1000: ribosomal 70S-EF-G complex at an authentic intermediate state dur...
Supramolecule | Name: ribosomal 70S-EF-G complex at an authentic intermediate state during translocation type: sample / ID: 1000 / Number unique components: 5 |
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Molecular weight | Experimental: 3 MDa / Theoretical: 3 MDa / Method: sedimentation |
-Supramolecule #1: 70S ribosome
Supramolecule | Name: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K-12 |
Molecular weight | Experimental: 3 MDa / Theoretical: 3 MDa |
-Macromolecule #1: Elongation Factor G
Macromolecule | Name: Elongation Factor G / type: protein_or_peptide / ID: 1 / Name.synonym: EF-G / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K-12 |
-Macromolecule #2: modified formyl-methionine specific initiator transfer RNA
Macromolecule | Name: modified formyl-methionine specific initiator transfer RNA type: rna / ID: 2 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K-12 |
-Macromolecule #3: formyl-methionine specific initiator transfer RNA
Macromolecule | Name: formyl-methionine specific initiator transfer RNA / type: rna / ID: 3 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K-12 |
-Macromolecule #4: messenger RNA
Macromolecule | Name: messenger RNA / type: rna / ID: 4 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K-12 |
-Experimental details
-Structure determination
Method | 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: 7.5 Details: 80 mM HEPES potassium, 75 mM NH4Cl, 10 mM MgCl2, 6 mM BME |
Grid | Details: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 96 K / Instrument: FEI VITROBOT MARK I / Method: blot for 5-10 seconds before plunging |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 39000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000 |
Sample stage | Specimen holder model: GATAN HELIUM |
Date | Jun 14, 2010 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.7 µm / Number real images: 308 / Average electron dose: 20 e/Å2 |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: each defocus group |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: OTHER / Software - Name: spider / Number images used: 279309 |
-Atomic model buiding 1
Initial model | PDB ID: 4kix |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-4v7b: |