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- EMDB-22467: 70S rotated ribosome stalled on truncated mRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-22467
Title70S rotated ribosome stalled on truncated mRNA
Map dataMap used in refinement for fit 2-V. Map was blocfiltered and no B factor was applied.
Sample
  • Complex: 70S rotated ribosome stalled on truncated mRNA
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsCarbone CE / Korostelev AA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107465 United States
CitationJournal: Nat Commun / Year: 2020
Title: ArfB can displace mRNA to rescue stalled ribosomes.
Authors: Christine E Carbone / Gabriel Demo / Rohini Madireddy / Egor Svidritskiy / Andrei A Korostelev /
Abstract: Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ...Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.
History
DepositionAug 16, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22467.png

Downloads & links

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Map

FileDownload / File: emd_22467.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap used in refinement for fit 2-V. Map was blocfiltered and no B factor was applied.
Voxel sizeX=Y=Z: 0.87 Å
Density
Contour LevelBy AUTHOR: 0.447 / Movie #1: 1
Minimum - Maximum-5.4598083 - 11.28592
Average (Standard dev.)0.09162398 (±0.58089024)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 389.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z448448448
origin x/y/z0.0000.0000.000
length x/y/z389.760389.760389.760
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS448448448
D min/max/mean-5.46011.2860.092

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Supplemental data

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Additional map: Original 3.4 angstrom map. No B factor applied.

Fileemd_22467_additional_1.map
AnnotationOriginal 3.4 angstrom map. No B factor applied.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_22467_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_22467_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 70S rotated ribosome stalled on truncated mRNA

EntireName: 70S rotated ribosome stalled on truncated mRNA
Components
  • Complex: 70S rotated ribosome stalled on truncated mRNA

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Supramolecule #1: 70S rotated ribosome stalled on truncated mRNA

SupramoleculeName: 70S rotated ribosome stalled on truncated mRNA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 30.5 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 48419

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