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- EMDB-18036: In situ structure of E. coli 70S ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-18036
TitleIn situ structure of E. coli 70S ribosome
Map data
Sample
  • Complex: In situ structure of E. coli 70S ribosome determined by subtomogram averaging
Keywordscryo-ET / subtomogram averaging / ribosome / 70S / E. coli
Biological speciesEscherichia coli BW25113 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 5.3 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_18036.png

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Map

FileDownload / File: emd_18036.map.gz / Format: CCP4 / Size: 96.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.697 Å
Density
Contour LevelBy AUTHOR: 0.001
Minimum - Maximum-0.0044297897 - 0.007945543
Average (Standard dev.)0.0000009470148 (±0.00025776718)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions294294294
Spacing294294294
CellA=B=C: 498.918 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18036_half_map_1.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Half map: #1

Fileemd_18036_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : In situ structure of E. coli 70S ribosome determined by subtomogr...

EntireName: In situ structure of E. coli 70S ribosome determined by subtomogram averaging
Components
  • Complex: In situ structure of E. coli 70S ribosome determined by subtomogram averaging

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Supramolecule #1: In situ structure of E. coli 70S ribosome determined by subtomogr...

SupramoleculeName: In situ structure of E. coli 70S ribosome determined by subtomogram averaging
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 2.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 6.5 / Details: LB medium
GridModel: Quantifoil / 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 / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 3.0 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: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 53000
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

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.0.9) / Number subtomograms used: 75670
ExtractionNumber tomograms: 104 / Number images used: 208000 / Reference model: 70S average derived from manual picking / Method: template matching / Software - Name: Warp (ver. 1.0.9)
Final 3D classificationSoftware - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)

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