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- EMDB-66749: In vitro structure of bacterial 50S ribosomes(CP) -

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Basic information

Entry
Database: EMDB / ID: EMD-66749
TitleIn vitro structure of bacterial 50S ribosomes(CP)
Map datafocused
Sample
  • Complex: In vitro E. coli ribosome sample
Keywordsribosome / 50S / in vitro / bacterial / CP
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsWu F / Naschberger A
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Commun Biol / Year: 2025
Title: In situ structure of bacterial 50S ribosomes at 3.0 Å resolution from vitreous sections.
Authors: Ashraf Al-Amoudi / Rozbeh Baradaran / Xukun Yuan / Fēi Wú / Andreas Naschberger /
Abstract: In situ high-resolution structure determination is limited to samples thin enough to be penetrated by the electron beam during imaging. Sample thinning involves focused ion or plasma beam milling of ...In situ high-resolution structure determination is limited to samples thin enough to be penetrated by the electron beam during imaging. Sample thinning involves focused ion or plasma beam milling of specimens to produce lamellae with thicknesses as low as 100-150 nm. However, surface damage caused by the milling process can extend 30-60 nm deep, restricting the usable lamella thickness. This imposes limitations on single-particle analysis of macromolecular complexes due to elevated structural noise, which cannot be avoided in situ because of the dense cellular environment. Alternative methods capable of producing thinner samples are needed to reduce background. Here, we demonstrate that high-resolution structures at side-chain level, free of orientation bias, can be obtained from vitreous sections prepared by cryo-ultramicrotomy, both in vitro and in situ. We optimized the method to produce sections as thin as ~40 nm, free from significant surface damage. Using this approach, we determined the structure of the 50S ribosomal subunit in vitro at 2.8 Å and in situ at 3 Å from bacterial cells. These results lay the foundation for future in situ studies of smaller complexes using CEMOVIS, as well as for methodological advances aimed at achieving compression-free sectioning.
History
DepositionOct 26, 2025-
Header (metadata) releaseFeb 4, 2026-
Map releaseFeb 4, 2026-
UpdateFeb 4, 2026-
Current statusFeb 4, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_66749.png

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Map

FileDownload / File: emd_66749.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 500 pix.
= 364.5 Å		0.73 Å/pix.
x 500 pix.
= 364.5 Å		0.73 Å/pix.
x 500 pix.
= 364.5 Å

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Voxel sizeX=Y=Z: 0.729 Å
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Contour LevelBy AUTHOR: 0.00392
Minimum - Maximum-0.014332241 - 0.019908912
Average (Standard dev.)-0.0002340587 (±0.00086958334)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 364.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_66749_msk_1.map
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Fileemd_66749_additional_1.map
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Half map: half

Fileemd_66749_half_map_1.map
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Half map: half

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Sample components

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Entire : In vitro E. coli ribosome sample

EntireName: In vitro E. coli ribosome sample
Components
  • Complex: In vitro E. coli ribosome sample

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Supramolecule #1: In vitro E. coli ribosome sample

SupramoleculeName: In vitro E. coli ribosome sample / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)

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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: 7.7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k 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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 81082
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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