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- EMDB-45638: Non-canonical E.coli 100S ribosome imaged in situ with rapid proc... -

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Basic information

Entry
Database: EMDB / ID: EMD-45638
TitleNon-canonical E.coli 100S ribosome imaged in situ with rapid processing pipeline
Map data
Sample
  • Complex: E.coli 100S pseudo-hibernating ribosome imaged in situ
Keywords100S / in situ / RIBOSOME / hibernating
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 15.0 Å
AuthorsPowell BM / Brant TS / Davis JH / Mosalaganti S
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM144542 United States
National Science Foundation (NSF, United States)CAREER-2046778 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32-GM007287 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM150019-01 United States
Citation
Journal: Commun Biol / Year: 2025
Title: Rapid structural analysis of bacterial ribosomes in situ.
Authors: Barrett M Powell / Tyler S Brant / Joseph H Davis / Shyamal Mosalaganti /
Abstract: Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data ...Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli, producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days and facilitating the discovery of a minor population of 100S-like disomes. We envision our approach to be widely applicable to related bacterial samples.
#1: Journal: bioRxiv / Year: 2024
Title: Rapid structural analysis of bacterial ribosomes
Authors: Powell BM / Brant TS / Davis JH / Mosalaganti S
History
DepositionJul 8, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45638.png

Downloads & links

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Map

FileDownload / File: emd_45638.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.84 Å/pix.
x 180 pix.
= 1231.2 Å		6.84 Å/pix.
x 180 pix.
= 1231.2 Å		6.84 Å/pix.
x 180 pix.
= 1231.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 6.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-3.6363676 - 9.148744000000001
Average (Standard dev.)0.009462161 (±0.5122655)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 1231.2001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45638_msk_1.map
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Half map: #2

Fileemd_45638_half_map_1.map
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Half map: #1

Fileemd_45638_half_map_2.map
Projections & Slices
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Sample components

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Entire : E.coli 100S pseudo-hibernating ribosome imaged in situ

EntireName: E.coli 100S pseudo-hibernating ribosome imaged in situ
Components
  • Complex: E.coli 100S pseudo-hibernating ribosome imaged in situ

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Supramolecule #1: E.coli 100S pseudo-hibernating ribosome imaged in situ

SupramoleculeName: E.coli 100S pseudo-hibernating ribosome imaged in situ
type: complex / ID: 1 / Parent: 0
Details: E. coli strain NCM3722 grown in LB at 37C to mid-log phase.
Source (natural)Organism: Escherichia coli (E. coli)

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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: 7.4
VitrificationCryogen name: ETHANE
DetailsCells grown to mid-log phase before pelleting and resuspended in LB to concentrate the cells 40-fold. Quantifoil R1/4 grids were glow-discharged for 30 seconds at 5 mA using an EasiGlow system (Pelco) one hour prior to sample application.

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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: 2.5 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: 2.9 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.4) / Number subtomograms used: 371
ExtractionNumber tomograms: 44 / Number images used: 44000 / Reference model: EMD-13270 / Method: Warp template matching / Software - Name: Warp (ver. 1.1.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.4)
FSC plot (resolution estimation)

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