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- EMDB-5386: tmRNA translocation and MLD-loading on the ribosome: a 70S-tmRNA-... -

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Basic information

Entry
Database: EMDB / ID: EMD-5386
TitletmRNA translocation and MLD-loading on the ribosome: a 70S-tmRNA-EF-G complex
Map dataOverall view of the 70S-tmRNA-SmpB-EF-G complex
Sample
  • Sample: Complex of 70S-tmRNA-SmpB-EF-G
  • Complex: Ribosome
Keywordstrans-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 / positive regulation of ribosome biogenesis / RNA-binding transcription regulator activity ...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 / positive regulation of ribosome biogenesis / RNA-binding transcription regulator activity / translational termination / 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 / 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
Ribosomal protein L11, bacterial-type / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 ...Ribosomal protein L11, bacterial-type / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / : / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L18, bacterial-type / : / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L5, bacterial-type / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / Ribosomal protein L36 superfamily / Ribosomal protein L36 / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L14P, bacterial-type / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L35 / Ribosomal protein L33 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L33 / Ribosomal L28 family / Ribosomal protein L33 superfamily / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L16 / Ribosomal protein S18, conserved site / Ribosomal protein L28/L24 / Ribosomal protein S18 signature. / Ribosomal protein L30, bacterial-type
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.3 Å
AuthorsRamrath 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
CitationJournal: 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
DepositionJan 20, 2012-
Header (metadata) releaseFeb 21, 2012-
Map releaseApr 30, 2012-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v6t
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5386_1.png

Downloads & links

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Map

FileDownload / File: emd_5386.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOverall view of the 70S-tmRNA-SmpB-EF-G complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 300 pix.
= 378. Å		1.26 Å/pix.
x 300 pix.
= 378. Å		1.26 Å/pix.
x 300 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-3.38914657 - 9.000638009999999
Average (Standard dev.)0.20524462 (±0.75120121)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.261.261.26
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-3.3899.0010.205

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Supplemental data

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Sample components

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Entire : Complex of 70S-tmRNA-SmpB-EF-G

EntireName: Complex of 70S-tmRNA-SmpB-EF-G
Components
  • Sample: Complex of 70S-tmRNA-SmpB-EF-G
  • Complex: Ribosome

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Supramolecule #1000: Complex of 70S-tmRNA-SmpB-EF-G

SupramoleculeName: 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
Molecular weightExperimental: 3 MDa / Theoretical: 3 MDa / Method: Sedimentation

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Supramolecule #1: Ribosome

SupramoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli) / Strain: CAN20
Molecular weightExperimental: 3 MDa / Theoretical: 3.05 MDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 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
StainingType: NEGATIVE / Details: flash frozen in liquid ethane
GridDetails: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 96.15 K / Instrument: OTHER / Details: Vitrification instrument: FEI VITROBOT / Method: blot for 10 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 77.15 K / Max: 81.15 K / Average: 77.15 K
DateDec 8, 2009
Image recordingCategory: 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder: Cartridge / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Defocus groups
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 68842

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Atomic model buiding 1

Initial modelPDB ID:

3r8s
PDB Unreleased entry

SoftwareName: Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-4v6t:
Structure of the bacterial ribosome complexed by tmRNA-SmpB and EF-G during translocation and MLD-loading

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