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- EMDB-36868: Cryo-EM structure of 30S ribosome with cleaved AP-mRNA bound comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-36868
TitleCryo-EM structure of 30S ribosome with cleaved AP-mRNA bound complex (Body 2)
Map data
Sample
  • Complex: 30S ribosome complex with AP-mRNA
Keywords30S Ribosome / AP-mRNA / RIBOSOME
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsRamachandran R / Afsar M / Shukla A
Funding support India, 3 items
OrganizationGrant numberCountry
Department of Science & Technology (DST, India)GAP0292 India
Council of Scientific & Industrial Research (CSIR)CII7032 & MLP0107 India
Department of Biotechnology (DBT, India)GAP0384 India
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Bacterial Rps3 counters oxidative and UV stress by recognizing and processing AP-sites on mRNA via a novel mechanism.
Authors: Mohammad Afsar / Ankita Shukla / Faiz Ali / Rahul Kumar Maurya / Suman Bharti / Nelam Kumar / Mohammad Sadik / Surabhi Chandra / Huma Rahil / Sanjay Kumar / Imran Ansari / Farheen Jahan / ...Authors: Mohammad Afsar / Ankita Shukla / Faiz Ali / Rahul Kumar Maurya / Suman Bharti / Nelam Kumar / Mohammad Sadik / Surabhi Chandra / Huma Rahil / Sanjay Kumar / Imran Ansari / Farheen Jahan / Saman Habib / Tanweer Hussain / Manju Yasoda Krishnan / Ravishankar Ramachandran /
Abstract: Lesions and stable secondary structures in mRNA severely impact the translation efficiency, causing ribosome stalling and collisions. Prokaryotic ribosomal proteins Rps3, Rps4 and Rps5, located in ...Lesions and stable secondary structures in mRNA severely impact the translation efficiency, causing ribosome stalling and collisions. Prokaryotic ribosomal proteins Rps3, Rps4 and Rps5, located in the mRNA entry tunnel, form the mRNA helicase center and unwind stable mRNA secondary structures during translation. However, the mechanism underlying the detection of lesions on translating mRNA is unclear. We used Cryo-EM, biochemical assays, and knockdown experiments to investigate the apurinic/apyrimidinic (AP) endoribonuclease activity of bacterial ribosomes on AP-site containing mRNA. Our biochemical assays show that Rps3, specifically the 130RR131 motif, is important for recognizing and performing the AP-endoribonuclease activity. Furthermore, structural analysis revealed cleaved mRNA product in the 30S ribosome entry tunnel. Additionally, knockdown studies in Mycobacterium tuberculosis confirmed the protective role of Rps3 against oxidative and UV stress. Overall, our results show that prokaryotic Rps3 recognizes and processes AP-sites on mRNA via a novel mechanism that is distinct from eukaryotes.
History
DepositionJul 17, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36868.png

Downloads & links

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Map

FileDownload / File: emd_36868.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 256 pix.
= 353.28 Å		1.38 Å/pix.
x 256 pix.
= 353.28 Å		1.38 Å/pix.
x 256 pix.
= 353.28 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.38 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.023265868 - 0.11297713
Average (Standard dev.)-0.00029008585 (±0.004869739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 353.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36868_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_36868_half_map_1.map
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Half map: #2

Fileemd_36868_half_map_2.map
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Sample components

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Entire : 30S ribosome complex with AP-mRNA

EntireName: 30S ribosome complex with AP-mRNA
Components
  • Complex: 30S ribosome complex with AP-mRNA

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Supramolecule #1: 30S ribosome complex with AP-mRNA

SupramoleculeName: 30S ribosome complex with AP-mRNA / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (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
GridMaterial: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 51.41 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7nat

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77582
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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