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- EMDB-18586: Cryo-EM reconstruction of crosslinked native Bacillus subtilis co... -

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Basic information

Entry
Database: EMDB / ID: EMD-18586
TitleCryo-EM reconstruction of crosslinked native Bacillus subtilis collided disome binding MutS2 SMR and KOW domains
Map dataCryo-EM reconstruction of B. subtilis MutS2-disome complex
Sample
  • Complex: MutS2-disome complex from B. subtilis, focus refined, stalled 70S
KeywordsMuts2 / collision / disome / splitting / quality control / RIBOSOME
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsPark E / Mackens-Kiani T / Berhane R / Esser H / Erdenebat C / Burroughs AM / Berninghausen O / Aravind L / Beckmann R / Green R / Buskirk AR
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)TRR174 Germany
CitationJournal: EMBO J / Year: 2024
Title: B. subtilis MutS2 splits stalled ribosomes into subunits without mRNA cleavage.
Authors: Esther N Park / Timur Mackens-Kiani / Rebekah Berhane / Hanna Esser / Chimeg Erdenebat / A Maxwell Burroughs / Otto Berninghausen / L Aravind / Roland Beckmann / Rachel Green / Allen R Buskirk /
Abstract: Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the ...Stalled ribosomes are rescued by pathways that recycle the ribosome and target the nascent polypeptide for degradation. In E. coli, these pathways are triggered by ribosome collisions through the recruitment of SmrB, a nuclease that cleaves the mRNA. In B. subtilis, the related protein MutS2 was recently implicated in ribosome rescue. Here we show that MutS2 is recruited to collisions by its SMR and KOW domains, and we reveal the interaction of these domains with collided ribosomes by cryo-EM. Using a combination of in vivo and in vitro approaches, we show that MutS2 uses its ABC ATPase activity to split ribosomes, targeting the nascent peptide for degradation through the ribosome quality control pathway. However, unlike SmrB, which cleaves mRNA in E. coli, we see no evidence that MutS2 mediates mRNA cleavage or promotes ribosome rescue by tmRNA. These findings clarify the biochemical and cellular roles of MutS2 in ribosome rescue in B. subtilis and raise questions about how these pathways function differently in diverse bacteria.
History
DepositionOct 3, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18586.png

Downloads & links

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Map

FileDownload / File: emd_18586.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of B. subtilis MutS2-disome complex
Voxel sizeX=Y=Z: 1.084 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.7113213 - 1.5576456
Average (Standard dev.)0.021356525 (±0.13419475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 487.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM reconstruction of B. subtilis MutS2-disome complex

Fileemd_18586_half_map_1.map
AnnotationCryo-EM reconstruction of B. subtilis MutS2-disome complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM reconstruction of B. subtilis MutS2-disome complex

Fileemd_18586_half_map_2.map
AnnotationCryo-EM reconstruction of B. subtilis MutS2-disome complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MutS2-disome complex from B. subtilis, focus refined, stalled 70S

EntireName: MutS2-disome complex from B. subtilis, focus refined, stalled 70S
Components
  • Complex: MutS2-disome complex from B. subtilis, focus refined, stalled 70S

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Supramolecule #1: MutS2-disome complex from B. subtilis, focus refined, stalled 70S

SupramoleculeName: MutS2-disome complex from B. subtilis, focus refined, stalled 70S
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6, #51, #53, #7-#50, #52
Source (natural)Organism: Bacillus subtilis (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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.4 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 19230
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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