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- EMDB-18585: Cryo-EM reconstruction of Bacillus subtilis collided disome bindi... -

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Basic information

Entry
Database: EMDB / ID: EMD-18585
TitleCryo-EM reconstruction of Bacillus subtilis collided disome binding MutS2 SMR and KOW domains
Map dataUnmasked refinement of MutS2-bound disome class
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: 3.8 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 17, 2024-
Map releaseJan 17, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18585.png

Downloads & links

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Map

FileDownload / File: emd_18585.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnmasked refinement of MutS2-bound disome class
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 500 pix.
= 522.5 Å		1.05 Å/pix.
x 500 pix.
= 522.5 Å		1.05 Å/pix.
x 500 pix.
= 522.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.275
Minimum - Maximum-0.49675846 - 1.5490619
Average (Standard dev.)0.0136791 (±0.097768255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 522.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Unmasked refinement of MutS2-bound disome class

Fileemd_18585_half_map_1.map
AnnotationUnmasked refinement of MutS2-bound disome class
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked refinement of MutS2-bound disome class

Fileemd_18585_half_map_2.map
AnnotationUnmasked refinement of MutS2-bound disome class
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.4 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11794
FSC plot (resolution estimation)

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