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- EMDB-16471: in vitro reconstituted yeast 40S ribosome complex with deubiquiti... -

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Basic information

Entry
Database: EMDB / ID: EMD-16471
Titlein vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
Map dataCryo-EM map of in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
Sample
  • Complex: in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsIkeuchi K / Buschauer R / Cheng J / Berninghausen O / Becker T / Beckmann R
Funding support Germany, Japan, 5 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB/TRR-174 Germany
German Research Foundation (DFG)BE1814/15-1 Germany
German Research Foundation (DFG)BE1814/1-1 Germany
Japan Society for the Promotion of Science (JSPS) Japan
Boehringer Ingelheim Fonds (BIF) Germany
CitationJournal: Nat Commun / Year: 2023
Title: Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2.
Authors: Ken Ikeuchi / Nives Ivic / Robert Buschauer / Jingdong Cheng / Thomas Fröhlich / Yoshitaka Matsuo / Otto Berninghausen / Toshifumi Inada / Thomas Becker / Roland Beckmann /
Abstract: In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its ...In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
History
DepositionJan 18, 2023-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateMay 24, 2023-
Current statusMay 24, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16471.png

Downloads & links

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Map

FileDownload / File: emd_16471.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 360 pix.
= 381.24 Å		1.06 Å/pix.
x 360 pix.
= 381.24 Å		1.06 Å/pix.
x 360 pix.
= 381.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.06749025 - 0.18461615
Average (Standard dev.)1.192801e-05 (±0.0039829407)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map B

Fileemd_16471_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_16471_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : in vitro reconstituted yeast 40S ribosome complex with deubiquiti...

EntireName: in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
Components
  • Complex: in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)

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Supramolecule #1: in vitro reconstituted yeast 40S ribosome complex with deubiquiti...

SupramoleculeName: in vitro reconstituted yeast 40S ribosome complex with deubiquitinating enzyme Otu2 bound to ubiquitinated eS7 - body 2 (40S head)
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#18
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303

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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: 46.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31646
Initial angle assignmentType: OTHER / Details: Relion
Final angle assignmentType: OTHER / Details: Relion
FSC plot (resolution estimation)

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