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- EMDB-47921: Control 50S ribosomal subunit for 50S subunit structures lacking ... -

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Basic information

Entry
Database: EMDB / ID: EMD-47921
TitleControl 50S ribosomal subunit for 50S subunit structures lacking the sarcin risin loop. Class 2.
Map data
Sample
  • Complex: Control 50S ribosomal subunit for 50S subunit structures lacking the sarcin risin loop. Class 2.
Keywords50S subunit Ribosome sarcin-ricin loop / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsOrtega J / Seffouh A
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-180305 Canada
CitationJournal: RNA / Year: 2025
Title: Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit.
Authors: Sepideh Fakhretaha Aval / Amal Seffouh / Kyung-Mee Moon / Leonard J Foster / Joaquin Ortega / Kurt Fredrick /
Abstract: The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are ...The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We find that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this activity is blocked in the absence of the SRL.
History
DepositionNov 14, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47921.png

Downloads & links

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Map

FileDownload / File: emd_47921.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 420 pix.
= 359.1 Å		0.86 Å/pix.
x 420 pix.
= 359.1 Å		0.86 Å/pix.
x 420 pix.
= 359.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.313
Minimum - Maximum-0.598568 - 1.9556286
Average (Standard dev.)0.01563479 (±0.088356934)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 359.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_47921_additional_1.map
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Half map: #1

Fileemd_47921_half_map_1.map
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Half map: #2

Fileemd_47921_half_map_2.map
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Sample components

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Entire : Control 50S ribosomal subunit for 50S subunit structures lacking ...

EntireName: Control 50S ribosomal subunit for 50S subunit structures lacking the sarcin risin loop. Class 2.
Components
  • Complex: Control 50S ribosomal subunit for 50S subunit structures lacking the sarcin risin loop. Class 2.

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Supramolecule #1: Control 50S ribosomal subunit for 50S subunit structures lacking ...

SupramoleculeName: Control 50S ribosomal subunit for 50S subunit structures lacking the sarcin risin loop. Class 2.
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 stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 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: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 239834
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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