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- EMDB-43942: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6... -

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Basic information

Entry
Database: EMDB / ID: EMD-43942
Title50S Assembly intermediate from SA22 strain with bL38 depleted uL6 over expression. Class 11
Map data
Sample
  • Complex: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6 over expression. Class 1
KeywordsRobosome / bL38 / uL6 / Escherichia coli / RIBOSOME
Biological speciesFlavobacterium johnsoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsOrtega J / Zhu H / Arpin D / Fredrick K
Funding support Canada, United States, 2 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2019-05799 Canada
National Science Foundation (NSF, United States)MCB-2029502 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Protein bL38 facilitates incorporation of uL6 during assembly of the 50S subunit in Flavobacterium johnsoniae.
Authors: Md Siddik Alom / Dominic Arpin / Haojun Zhu / Brenna N Hay / Leonard J Foster / Joaquin Ortega / Kurt Fredrick /
Abstract: Previous studies of the 70S ribosome from Flavobacterium johnsoniae revealed a novel ribosomal protein, bL38, which interacts with uL6 on the 50S subunit. This 5.6 kDa protein is conserved across the ...Previous studies of the 70S ribosome from Flavobacterium johnsoniae revealed a novel ribosomal protein, bL38, which interacts with uL6 on the 50S subunit. This 5.6 kDa protein is conserved across the Bacteroidia and encoded downstream of bL28 and bL33 in a three-gene operon. Here, we show that bL38 is critical for the growth of F. johnsoniae, and depletion of bL38 leads to accumulation of immature 50S particles, which lack uL6 and retain precursor rRNA sequences. Cryo-EM analysis of these particles reveals several putative assembly intermediates, all showing an absence of electron density for uL6 and the entire uL12 stalk region and additional densities corresponding to the unprocessed ends of the pre-23S rRNA. Extra copies of the uL6 gene can rescue the phenotypes caused by bL38 depletion, suggesting that bL38 facilitates uL6 incorporation during 50S subunit biogenesis. Cryo-EM analysis of 50S particles from this rescued strain reveals nearly twice as many intermediates, suggesting a broader and more robust assembly landscape. Differential scanning fluorimetry shows that uL6 of F. johnsoniae is intrinsically unstable, and bL38 increases the melting temperature of uL6 by 12°C. Collectively, these data suggest that bL38 binds and stabilizes uL6, thereby promoting 50S biogenesis in the Bacteroidia.
History
DepositionMar 6, 2024-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43942.png

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Map

FileDownload / File: emd_43942.map.gz / Format: CCP4 / Size: 290.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 424 pix.
= 362.52 Å		0.86 Å/pix.
x 424 pix.
= 362.52 Å		0.86 Å/pix.
x 424 pix.
= 362.52 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.093
Minimum - Maximum-0.10335426 - 0.32897592
Average (Standard dev.)0.0018746862 (±0.021268347)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions424424424
Spacing424424424
CellA=B=C: 362.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_43942_additional_1.map
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Half map: #1

Fileemd_43942_half_map_1.map
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Half map: #2

Fileemd_43942_half_map_2.map
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Sample components

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Entire : 50S Assembly intermediate from SA22 strain with bL38 depleted uL6...

EntireName: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6 over expression. Class 1
Components
  • Complex: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6 over expression. Class 1

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Supramolecule #1: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6...

SupramoleculeName: 50S Assembly intermediate from SA22 strain with bL38 depleted uL6 over expression. Class 1
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Flavobacterium johnsoniae (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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.225 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11136
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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