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- EMDB-43853: Structure of Circularly Permuted 50S Ribosomal Subunit Assembly I... -

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Basic information

Entry
Database: EMDB / ID: EMD-43853
TitleStructure of Circularly Permuted 50S Ribosomal Subunit Assembly Intermediate - CP63 Class D
Map dataClass D CP63 50S Ribosomal Intermediate
Sample
  • Complex: CP63 iSAT 50S ribosomal subunit assembly intermediate
Keywords50S subunit / assembly intermediate / RNA-protein complex / ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsDong X / Sheng K / Williamson JR
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136412 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM053757 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI170855 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Assembly of the bacterial ribosome with circularly permuted rRNA.
Authors: Xiyu Dong / Kai Sheng / Luca F R Gebert / Sriram Aiyer / Ian J MacRae / Dmitry Lyumkis / James R Williamson /
Abstract: Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of ...Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the structural insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of 23 resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.
History
DepositionFeb 28, 2024-
Header (metadata) releaseMar 20, 2024-
Map releaseMar 20, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43853.png

Downloads & links

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Map

FileDownload / File: emd_43853.map.gz / Format: CCP4 / Size: 187 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClass D CP63 50S Ribosomal Intermediate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.1.151.151.15
CCP4 map header1.881.881.88
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.17410634 - 0.84887433
Average (Standard dev.)-0.00054454734 (±0.050125793)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions366366366
Spacing366366366
CellA=B=C: 420.9 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Class D CP63 50S Ribosomal Intermediate - Half Map 2

Fileemd_43853_half_map_1.map
AnnotationClass D CP63 50S Ribosomal Intermediate - Half Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CP63 iSAT 50S ribosomal subunit assembly intermediate

EntireName: CP63 iSAT 50S ribosomal subunit assembly intermediate
Components
  • Complex: CP63 iSAT 50S ribosomal subunit assembly intermediate

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Supramolecule #1: CP63 iSAT 50S ribosomal subunit assembly intermediate

SupramoleculeName: CP63 iSAT 50S ribosomal subunit assembly intermediate / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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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:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris Hydrochloride
100.0 mMNH4ClAmmonium Chloride
10.0 mMMgCl2Magnesium Chloride
0.5 mMC10H16N2O8Ethylenediaminetetraacetic Acid
6.0 mMC2H6OSBeta-mercaptoethanol

Details: Most of the sucrose was removed by spin concentration.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 3 microliter of the sample was added..
DetailsThe in vitro assembled large ribosomal subunit was purified by sucrose gradient and was spin-concentrated in a 100 kDa MW-cutoff filter.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
DetailsIn order to account for highly preferred orientation of the specimen, data were acquired using tilts ranging at -20 degrees.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2277 / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 686910 / Details: Particles were selected by cryoSPARC.
Startup modelType of model: NONE / Details: Ab initio reconstruction in CryoSPARC was used.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 8020
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Details: Non-uniform Refinement of cryoSPARC was used.
Final 3D classificationNumber classes: 10 / Avg.num./class: 6000 / Details: 3D classification of cryoSPARC was used.
FSC plot (resolution estimation)

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