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- PDB-4v6t: Structure of the bacterial ribosome complexed by tmRNA-SmpB and E... -
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Basic information
Entry | Database: PDB / ID: 4v6t | ||||||||||||
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Title | Structure of the bacterial ribosome complexed by tmRNA-SmpB and EF-G during translocation and MLD-loading | ||||||||||||
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![]() | RIBOSOME / trans-translation / MLD-loading / translocation | ||||||||||||
Function / homology | ![]() stringent response / 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 ...stringent response / 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 / translational termination / 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 / 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: 8.3 Å | ||||||||||||
![]() | Ramrath, D.J.F. / Yamamoto, H. / Rother, K. / Wittek, D. / Pech, M. / Mielke, T. / Loerke, J. / Scheerer, P. / Ivanov, P. / Teraoka, Y. ...Ramrath, D.J.F. / Yamamoto, H. / Rother, K. / Wittek, D. / Pech, M. / Mielke, T. / Loerke, J. / Scheerer, P. / Ivanov, P. / Teraoka, Y. / Shpanchenko, O. / Nierhaus, K.H. / Spahn, C.M.T. | ||||||||||||
![]() | ![]() 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
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 3.5 MB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 3 MB | Display | |
Data in XML | ![]() | 458.5 KB | Display | |
Data in CIF | ![]() | 689.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5386MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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1 | ![]()
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Components
-RNA chain , 5 types, 5 molecules AAAVAXBABB
#1: RNA chain | Mass: 498725.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#22: RNA chain | Mass: 117212.297 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#24: RNA chain | Mass: 24802.785 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#26: RNA chain | Mass: 941306.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#56: RNA chain | Mass: 38483.926 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: 24253.943 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#3: Protein | Mass: 23078.785 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#4: Protein | Mass: 23383.002 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#5: Protein | Mass: 15804.282 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#6: Protein | Mass: 11669.371 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#7: Protein | Mass: 16861.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#8: Protein | Mass: 14015.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#9: Protein | Mass: 14554.882 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#10: Protein | Mass: 11196.988 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#11: Protein | Mass: 12487.200 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#12: Protein | Mass: 13636.961 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#13: Protein | Mass: 12625.753 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#14: Protein | Mass: 11475.364 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#15: Protein | Mass: 10159.621 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: 9263.946 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#18: Protein | Mass: 6466.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#19: Protein | Mass: 9057.626 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#20: Protein | Mass: 9506.190 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#21: Protein | Mass: 6067.081 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-Protein , 2 types, 2 molecules AWAY
#23: Protein | Mass: 14177.479 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#25: Protein | Mass: 76977.102 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
+50S ribosomal protein ... , 29 types, 29 molecules BCBDBEBFBGBHBIBJBKBLBMBNBOBPBQBRBSBTBUBVBWBXBYBZB0B1B2B3B4
-Details
Sequence details | MODELED SEQUENCES FOR SSRA_BINDING PROTEIN (UNP Q8RR57 RESIDUES 1-123) AND ELONGATION FACTOR G (UNP ...MODELED SEQUENCES FOR SSRA_BINDING PROTEIN (UNP Q8RR57 RESIDUES 1-123) AND ELONGATION |
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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
Component | Name: Ribosomal complex 70S-tmRNA-SmpB-tRNA-EFG-GDP-FA / Type: RIBOSOME / Details: in vitro reconstituted ribosomal complex |
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Buffer solution | pH: 7.6 |
Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: Quantifoil R3-3 Cu mesh with 2 nm carbon support film |
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 96.15 K / Humidity: 100 % Details: blot for 5-10 seconds before plunging into liquid ethane (FEI Vitrobot) Method: blot for 5-10 seconds before plunging |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 / Date: Dec 8, 2009 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 39000 X / Calibrated magnification: 39000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 2500 nm / Cs: 2 mm |
Specimen holder | Temperature: 77 K |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM |
Image scans | Num. digital images: 302 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Relative weight: 1 |
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Processing
EM software |
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CTF correction | Details: The volumes were CTF-corrected in defocus groups, with an average of approximately 215 individual images per group | ||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||
3D reconstruction | Method: angular refinement / Resolution: 8.3 Å / Num. of particles: 68843 / Nominal pixel size: 1.26 Å / Actual pixel size: 1.26 Å / Symmetry type: POINT | ||||||||||||
Refinement step | Cycle: LAST
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