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- EMDB-44152: E.coli 70S ribosome imaged in situ with rapid processing pipeline -

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Basic information

Entry
Database: EMDB / ID: EMD-44152
TitleE.coli 70S ribosome imaged in situ with rapid processing pipeline
Map dataprimary map which was automatically filtered and sharpened by M during refinement
Sample
  • Cell: E.coli 70S ribosome imaged in situ
Keywords70S ribosome / in situ / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 5.88 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
CitationJournal: bioRxiv / Year: 2024
Title: Rapid structural analysis of bacterial ribosomes .
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 the development of a rapid 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 , producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days, and we expect this workflow will be widely applicable to related bacterial samples.
History
DepositionMar 20, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateFeb 12, 2025-
Current statusFeb 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44152.png

Downloads & links

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Map

FileDownload / File: emd_44152.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map which was automatically filtered and sharpened by M during refinement
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 300 pix.
= 600. Å		2 Å/pix.
x 300 pix.
= 600. Å		2 Å/pix.
x 300 pix.
= 600. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0026
Minimum - Maximum-0.0065673804 - 0.01599694
Average (Standard dev.)0.000007909148 (±0.0008722391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 600.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44152_msk_1.map
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Additional map: supplementary map which was denoised by M during refinement

Fileemd_44152_additional_1.map
Annotationsupplementary map which was denoised by M during refinement
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Additional map: supplementary local resolution map whcih was generated by...

Fileemd_44152_additional_2.map
Annotationsupplementary local resolution map whcih was generated by M during refinement
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Half map: half-map 2 reconstructed by M

Fileemd_44152_half_map_1.map
Annotationhalf-map 2 reconstructed by M
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AxesZYX

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Half map: half-map 1 reconstructed by M

Fileemd_44152_half_map_2.map
Annotationhalf-map 1 reconstructed by M
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AxesZYX

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Sample components

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Entire : E.coli 70S ribosome imaged in situ

EntireName: E.coli 70S ribosome imaged in situ
Components
  • Cell: E.coli 70S ribosome imaged in situ

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Supramolecule #1: E.coli 70S ribosome imaged in situ

SupramoleculeName: E.coli 70S ribosome imaged in situ / type: cell / 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
GridMaterial: COPPER / Mesh: 200
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: 5.88 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.0.9)
Software - details: M was used to reconstruct, filter, and sharpen the final map
Number subtomograms used: 8170
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: Warp (ver. 1.0.9)
Software - details: M was used to fine-tune angles derived from RELION 3-D autorefine
FSC plot (resolution estimation)

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