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- EMDB-1311: Cryo-EM visualization of transfer messenger RNA with two SmpBs in... -

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Basic information

Entry
Database: EMDB / ID: EMD-1311
TitleCryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome.
Map dataCryo-EM map of Thermus thermophilus 70S ribosome bound with tmRNA, two small protein B, and EF-Tu in the presence of S1 in solution.
Sample
  • Sample: Thermus thermophilus 70S ribosome
  • Complex: 70S RibosomeRibosome
  • Protein or peptide: small protein B
  • Protein or peptide: EF-Tu
  • RNA: tmRNATransfer-messenger RNA
  • RNA: tRNATransfer RNA
Function / homologytrans-translation / SsrA-binding protein / SsrA-binding protein, conserved site / Small protein B / SmpB protein / SsrA-binding protein. / RNA binding / cytosol / SsrA-binding protein
Function and homology information
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 13.6 Å
AuthorsKaur S / Gillet R / Li W / Gursky R / Frank J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2006
Title: Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome.
Authors: Sukhjit Kaur / Reynald Gillet / Wen Li / Richard Gursky / Joachim Frank /
Abstract: In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer ...In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer messenger RNA (tmRNA), to rescue such a stalled system. tmRNA contains a transfer RNA (tRNA)-like domain (TLD), which enters the ribosome as a tRNA and places an ORF into the mRNA channel. This ORF codes for a signal marking the polypeptide for degradation and ends in a stop codon, leading to release of the faulty polypeptide and recycling of the ribosome. The binding of tmRNA to the stalled ribosome is mediated by small protein B (SmpB). By means of cryo-EM, we obtained a density map for the preaccommodated state of the tmRNA.SmpB.EF-Tu.70S ribosome complex with much improved definition for the tmRNA-SmpB complex, showing two SmpB molecules bound per ribosome, one toward the A site on the 30S subunit side and the other bound to the 50S subunit near the GTPase-associated center. tmRNA is strongly attached to the 30S subunit head by multiple contact sites, involving most of its pseudoknots and helices. The map clarifies that the TLD is located near helix 34 and protein S19 of the 30S subunit, rather than in the A site as tRNA for normal translation, so that the TLD is oriented toward the ORF.
History
DepositionDec 13, 2006-
Header (metadata) releaseDec 14, 2006-
Map releaseDec 14, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 30.915807559
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 30.915807559
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2ob7
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1311.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Thermus thermophilus 70S ribosome bound with tmRNA, two small protein B, and EF-Tu in the presence of S1 in solution.
Voxel sizeX=Y=Z: 2.82 Å
Density
Contour Level1: 73.200000000000003 / Movie #1: 30.9158076
Minimum - Maximum-117.853999999999999 - 304.088000000000022
Average (Standard dev.)6.06499 (±30.402000000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z366.600366.600366.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-63-63-63
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-117.854304.0886.065

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Supplemental data

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Sample components

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Entire : Thermus thermophilus 70S ribosome

EntireName: Thermus thermophilus 70S ribosome
Components
  • Sample: Thermus thermophilus 70S ribosome
  • Complex: 70S RibosomeRibosome
  • Protein or peptide: small protein B
  • Protein or peptide: EF-Tu
  • RNA: tmRNATransfer-messenger RNA
  • RNA: tRNATransfer RNA

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Supramolecule #1000: Thermus thermophilus 70S ribosome

SupramoleculeName: Thermus thermophilus 70S ribosome / type: sample / ID: 1000 / Number unique components: 5
Molecular weightTheoretical: 2.4 MDa

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Supramolecule #1: 70S Ribosome

SupramoleculeName: 70S Ribosome / type: complex / ID: 1 / Ribosome-details: ribosome-prokaryote: ALL
Molecular weightExperimental: 2.3 MDa

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Macromolecule #1: small protein B

MacromoleculeName: small protein B / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightExperimental: 19 KDa

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Macromolecule #4: EF-Tu

MacromoleculeName: EF-Tu / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus (bacteria)

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Macromolecule #2: tmRNA

MacromoleculeName: tmRNA / type: rna / ID: 2 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Thermus thermophilus (bacteria)

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Macromolecule #3: tRNA

MacromoleculeName: tRNA / type: rna / ID: 3 / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Thermus thermophilus (bacteria) / synonym: tRNA

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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

BufferpH: 7.5 / Details: see Methods
StainingType: NEGATIVE / Details: No staining (Cryo-EM)
GridDetails: Quanti-foil grids coated with a thin carbon layer
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 279 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blot for 5 seconds before plunging Rapid plunge freezing in liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49696 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.635 µm / Nominal defocus min: 1.475 µm / Nominal magnification: 5000
Sample stageSpecimen holder: cryo transfer / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 93 K / Average: 93 K
Alignment procedureLegacy - Electron beam tilt params: 0
DateAug 13, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 27 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correctionn of 3D map
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER package / Number images used: 52829

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Atomic model buiding 1

SoftwareName: O
DetailsProtocol: Rigid Body. manual fitting with O
RefinementProtocol: RIGID BODY FIT
Output model

PDB-2ob7:
Structure of tmRNA-(SmpB)2 complex as inferred from cryo-EM

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