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- EMDB-29842: Time-resolved cryo-EM study of the 70S recycling by the HflX:3rd ... -

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Entry
Database: EMDB / ID: EMD-29842
TitleTime-resolved cryo-EM study of the 70S recycling by the HflX:3rd Intermediate, 50S focused and 30S subtracted
Map dataTime-resolved cryo-EM study of the 70S recycling by the HflX:1st intermediate, 50S focused and 30S subtracted
Sample
  • Complex: Apo 70S 1st intermediate
KeywordsRecycling / Time-resolved Cryo-EM / 70S / HflX / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsBhattacharjee S / Brown PZ / Frank J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139453 United States
CitationJournal: Cell / Year: 2024
Title: Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.
Authors: Sayan Bhattacharjee / Xiangsong Feng / Suvrajit Maji / Prikshat Dadhwal / Zhening Zhang / Zuben P Brown / Joachim Frank /
Abstract: The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real ...The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.
History
DepositionFeb 17, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29842.png

Downloads & links

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Map

FileDownload / File: emd_29842.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTime-resolved cryo-EM study of the 70S recycling by the HflX:1st intermediate, 50S focused and 30S subtracted
Voxel sizeX=Y=Z: 1.02529 Å
Density
Contour LevelBy AUTHOR: 0.123
Minimum - Maximum-0.33644366 - 0.59820825
Average (Standard dev.)0.0044008037 (±0.028635401)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 348.5986 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29842_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Time-resolved cryo-EM study of the 70S recycling by...

Fileemd_29842_half_map_1.map
AnnotationTime-resolved cryo-EM study of the 70S recycling by the HflX:1st intermediate, 50S focused and 30S subtracted, half-I
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Time-resolved cryo-EM study of the 70S recycling by...

Fileemd_29842_half_map_2.map
AnnotationTime-resolved cryo-EM study of the 70S recycling by the HflX:1st intermediate, 50S focused and 30S subtracted, half-II
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apo 70S 1st intermediate

EntireName: Apo 70S 1st intermediate
Components
  • Complex: Apo 70S 1st intermediate

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Supramolecule #1: Apo 70S 1st intermediate

SupramoleculeName: Apo 70S 1st intermediate / 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 statecell

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 113038
DetailsHigh-pass filtered
FSC plot (resolution estimation)

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