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- EMDB-29042: Structure of iSAT large ribosomal subunit assembly intermediate -... -

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Basic information

Entry
Database: EMDB / ID: EMD-29042
TitleStructure of iSAT large ribosomal subunit assembly intermediate - Class B2
Map dataClass B2 iSAT 50S Ribosomal Intermediate
Sample
  • Complex: iSAT large ribosomal subunit assembly intermediate (Class B2)
Keywords50S subunit / assembly intermediate / in vitro / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsDong X / Doerfel LK / Sheng K / Rabuck-Gibbons JN / Popova AM / Lyumkis D / 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: 2023
Title: Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.
Authors: Xiyu Dong / Lili K Doerfel / Kai Sheng / Jessica N Rabuck-Gibbons / Anna M Popova / Dmitry Lyumkis / James R Williamson /
Abstract: Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental ...Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.
History
DepositionDec 8, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29042.png

Downloads & links

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Map

FileDownload / File: emd_29042.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClass B2 iSAT 50S Ribosomal Intermediate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.1 Å/pix.
x 160 pix.
= 335.36 Å		2.1 Å/pix.
x 160 pix.
= 335.36 Å		2.1 Å/pix.
x 160 pix.
= 335.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.096 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-0.61890763 - 3.2407963
Average (Standard dev.)0.02297961 (±0.19315074)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Class B2 iSAT 50S Ribosomal Intermediate - Half Map 1

Fileemd_29042_half_map_1.map
AnnotationClass B2 iSAT 50S Ribosomal Intermediate - Half Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Class B2 iSAT 50S Ribosomal Intermediate - Half Map 2

Fileemd_29042_half_map_2.map
AnnotationClass B2 iSAT 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 : iSAT large ribosomal subunit assembly intermediate (Class B2)

EntireName: iSAT large ribosomal subunit assembly intermediate (Class B2)
Components
  • Complex: iSAT large ribosomal subunit assembly intermediate (Class B2)

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Supramolecule #1: iSAT large ribosomal subunit assembly intermediate (Class B2)

SupramoleculeName: iSAT large ribosomal subunit assembly intermediate (Class B2)
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.
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / 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 TITAN KRIOS
DetailsIn order to account for highly preferred orientation of the specimen, data were acquired using tilts ranging from 10-60 degrees, in addition to untilted images at 0 degrees. The CTF was estimated on a per-particle basis to account for the gradient of CTF values across individual micrographs
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number real images: 4607 / Average electron dose: 50.0 e/Å2
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 / 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

Particle selectionNumber selected: 1120554 / Details: Particles were selected by cryoSPARC.
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model / Details: Ab initio model was generated in CryoSPARC.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 8581
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Ab initio reconstruction in CryoSPARC was used.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Homogeneous refinement in CryoSPARC was used.
FSC plot (resolution estimation)

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