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- EMDB-22471: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in th... -

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Basic information

Entry
Database: EMDB / ID: EMD-22471
TitleRotated 70S ribosome stalled on long mRNA with ArfB-1 bound in the A site (+9-V)
Map dataRefinement map for fit 9-V with B factor softened 25 angstroms squared.
Sample
  • Complex: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in the A site (+9-V)
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 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)R01GM107465 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127094 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 17, 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: 1.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22471.png

Downloads & links

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Map

FileDownload / File: emd_22471.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefinement map for fit 9-V with B factor softened 25 angstroms squared.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 360 pix.
= 375.12 Å		1.04 Å/pix.
x 360 pix.
= 375.12 Å		1.04 Å/pix.
x 360 pix.
= 375.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.042 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.06 / Movie #1: 1.4
Minimum - Maximum-3.1216626 - 10.858418
Average (Standard dev.)0.012000945 (±0.7930609)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 375.12003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0421.0421.042
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z375.120375.120375.120
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-3.12210.8580.012

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Supplemental data

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Additional map: Original 3.3 angstrom map with no B factor applied.

Fileemd_22471_additional_1.map
AnnotationOriginal 3.3 angstrom map with 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 1

Fileemd_22471_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_22471_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in th...

EntireName: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in the A site (+9-V)
Components
  • Complex: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in the A site (+9-V)

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Supramolecule #1: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in th...

SupramoleculeName: Rotated 70S ribosome stalled on long mRNA with ArfB-1 bound in the A site (+9-V)
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12969
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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