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- EMDB-18042: E. coli ED1a 70S ribosome bound to mRNA A-tRNA, P-tRNA, E-tRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-18042
TitleE. coli ED1a 70S ribosome bound to mRNA A-tRNA, P-tRNA, E-tRNA
Map data
Sample
  • Complex: Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a determined by single particle cryo-EM
Keywordscryo-EM / ribosome / 70S / E. coli
Biological speciesEscherichia coli ED1a (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsKhusainov I / Romanov N / Goemans C / Turonova B / Zimmerli CE / Welsch S / Langer JD / Typas A / Beck M
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2024
Title: Bactericidal effect of tetracycline in E. coli strain ED1a may be associated with ribosome dysfunction.
Authors: Iskander Khusainov / Natalie Romanov / Camille Goemans / Beata Turoňová / Christian E Zimmerli / Sonja Welsch / Julian D Langer / Athanasios Typas / Martin Beck /
Abstract: Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal ...Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.
History
DepositionJul 27, 2023-
Header (metadata) releaseJun 19, 2024-
Map releaseJun 19, 2024-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18042.png

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Map

FileDownload / File: emd_18042.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 400 pix.
= 502.2 Å		1.26 Å/pix.
x 400 pix.
= 502.2 Å		1.26 Å/pix.
x 400 pix.
= 502.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2555 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.304325 - 3.6243203
Average (Standard dev.)0.0002099393 (±0.12051877)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 502.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18042_msk_1.map
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Half map: #1

Fileemd_18042_half_map_1.map
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Half map: #2

Fileemd_18042_half_map_2.map
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Sample components

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Entire : Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a...

EntireName: Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a determined by single particle cryo-EM
Components
  • Complex: Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a determined by single particle cryo-EM

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Supramolecule #1: Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a...

SupramoleculeName: Structure of 70S-mRNA-APE-tRNA ribosome complex from E. coli ED1a determined by single particle cryo-EM
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli ED1a (bacteria)
Molecular weightTheoretical: 2.5 MDa

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
0.01 MHepes-KOH
0.06 MPotassium chlorideKCl
0.015 MMagnesium acetateMgCH3COOCH3COO
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.25 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1211457
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 132290
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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