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- EMDB-16548: Local refinement map of Otu2 C-terminal domain bound to ubiquitin... -

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Basic information

Entry
Database: EMDB / ID: EMD-16548
TitleLocal refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
Map dataLocal refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
Sample
  • Complex: Local refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
    • Protein or peptide: OTU domain-containing protein 2 (Otu2) C178S
    • Protein or peptide: Ubiquitin
    • Protein or peptide: 40S ribosomal protein S7-A
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae W303 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 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)JSPS Overseas Research Fellowships Japan
Boehringer Ingelheim Fonds (BIF)PhD fellowship 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 25, 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_16548.png

Downloads & links

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Map

FileDownload / File: emd_16548.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 420 pix.
= 438.9 Å		1.05 Å/pix.
x 420 pix.
= 438.9 Å		1.05 Å/pix.
x 420 pix.
= 438.9 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: 0.4
Minimum - Maximum-4.063359 - 7.3144956
Average (Standard dev.)0.0017822396 (±0.06535153)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 438.9 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map A

Fileemd_16548_half_map_1.map
AnnotationHalf-map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map B

Fileemd_16548_half_map_2.map
AnnotationHalf-map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Local refinement map of Otu2 C-terminal domain bound to ubiquitin...

EntireName: Local refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
Components
  • Complex: Local refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
    • Protein or peptide: OTU domain-containing protein 2 (Otu2) C178S
    • Protein or peptide: Ubiquitin
    • Protein or peptide: 40S ribosomal protein S7-A

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Supramolecule #1: Local refinement map of Otu2 C-terminal domain bound to ubiquitin...

SupramoleculeName: Local refinement map of Otu2 C-terminal domain bound to ubiquitinated eS7 on yeast 40S ribosome
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: W303

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Macromolecule #1: OTU domain-containing protein 2 (Otu2) C178S

MacromoleculeName: OTU domain-containing protein 2 (Otu2) C178S / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae W303 (yeast)
SequenceString: MTGMESGENL ENMEDILARH RKENKDLQNK ITGMKKQATK SKRKEVNSKC LDLQDKLKTK QENEIRDWKI ANNEVFDAEQ EDEVTPEKL LEQLSISRDE KEQQNVPVQQ QQQGQTKKRR NRQKERLAKR DAAIAKMKEE AALEASKQPD LKKMEQESID Q LCELKKLK ...String:
MTGMESGENL ENMEDILARH RKENKDLQNK ITGMKKQATK SKRKEVNSKC LDLQDKLKTK QENEIRDWKI ANNEVFDAEQ EDEVTPEKL LEQLSISRDE KEQQNVPVQQ QQQGQTKKRR NRQKERLAKR DAAIAKMKEE AALEASKQPD LKKMEQESID Q LCELKKLK QFDIQPDGHS LFASILDQLK LRHDPKKLDQ DMDVMKLRWL SCNYVQEHRD DFIPYLFDEE TMKMKDIDEY TK EMEHTAQ WGGEIEILAL SHVFDCPISI LMSGRPIQVY NECGKNPELK LVYYKHSYAL GEHYNSLHDS

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Macromolecule #2: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
SequenceString:
MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGG

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Macromolecule #3: 40S ribosomal protein S7-A

MacromoleculeName: 40S ribosomal protein S7-A / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
SequenceString:
MSAPQAKILS QAPTELELQV AQAFVELENS SPELKAELRP LQFKSIREID VAGGKKALAI FVPVPSLAGF HKVQTKLTRE LEKKFQDRHV IFLAERRILP KPSRTSRQVQ KRPRSRTLTA VHDKILEDLV FPTEIVGKRV RYLVGGNKIQ KVLLDSKDVQ QIDYKLESFQ AVYNKLTGKQ IVFEIPSETH

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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) / Detector mode: COUNTING / Average electron dose: 46.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 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: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31008
Initial angle assignmentType: OTHER / Details: Relion
Final angle assignmentType: OTHER / Details: Relion
FSC plot (resolution estimation)

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