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- EMDB-40532: E. coli 50S intermediate, deaD deletion strain, class: dead-C-a1 -

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Basic information

Entry
Database: EMDB / ID: EMD-40532
TitleE. coli 50S intermediate, deaD deletion strain, class: dead-C-a1
Map datadead-C-a1 class Particle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by CryoSPARC ab-initio subclassification. Aligned to 50S ref and resample to same grid.
Sample
  • Complex: E. coli 50S assembly intermediate from deaD-deletion strain, grown at 19 degrees Celsius
KeywordsRibosome / 50S intermediate / RNP
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.14 Å
AuthorsSheng K / Williamson JR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136412 United States
CitationJournal: Nat Commun / Year: 2023
Title: Assembly landscape for the bacterial large ribosomal subunit.
Authors: Kai Sheng / Ning Li / Jessica N Rabuck-Gibbons / Xiyu Dong / Dmitry Lyumkis / James R Williamson /
Abstract: Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic ...Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.
History
DepositionApr 17, 2023-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateSep 27, 2023-
Current statusSep 27, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40532.png

Downloads & links

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Map

FileDownload / File: emd_40532.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdead-C-a1 class Particle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by CryoSPARC ab-initio subclassification. Aligned to 50S ref and resample to same grid.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-1.3999804 - 3.7899547
Average (Standard dev.)0.0008017669 (±0.20619178)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: dead-C-a1 classParticle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by...

Fileemd_40532_half_map_1.map
Annotationdead-C-a1 classParticle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by CryoSPARC ab-initio subclassification.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: dead-C-a1 classParticle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by...

Fileemd_40532_half_map_2.map
Annotationdead-C-a1 classParticle stack from (https://doi.org/10.1101/2022.11.09.515851) was analyzed by CryoSPARC ab-initio subclassification.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : E. coli 50S assembly intermediate from deaD-deletion strain, grow...

EntireName: E. coli 50S assembly intermediate from deaD-deletion strain, grown at 19 degrees Celsius
Components
  • Complex: E. coli 50S assembly intermediate from deaD-deletion strain, grown at 19 degrees Celsius

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Supramolecule #1: E. coli 50S assembly intermediate from deaD-deletion strain, grow...

SupramoleculeName: E. coli 50S assembly intermediate from deaD-deletion strain, grown at 19 degrees Celsius
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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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: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
100.0 mMammonium chlorideNH4Cl
10.0 mMmagnesium chlorideMgCl2
0.5 mMEDTA
6.0 mMbeta-mercaptoethanol
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 33.0 e/Å2
Details: Data were collected with tilts ranging from 0 to 60 degrees at 10-degree increments.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
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

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2028
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Homogeneous refinement in CryoSPARC was used.
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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