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- EMDB-8446: Structure of bL17-depleted large ribosomal subunit assembly inter... -

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Entry
Database: EMDB / ID: 8446
TitleStructure of bL17-depleted large ribosomal subunit assembly intermediate - Class D1
Map dataClass D1 bL17-Depleted 50S Ribosomal Biogenesis Intermediate
SamplebL17-depleted large ribosomal subunit assembly intermediate - Class D1:
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / 4.7 Å resolution
AuthorsTan Y / Davis JH / Lyumkis D
CitationJournal: Cell / Year: 2016
Title: Modular Assembly of the Bacterial Large Ribosomal Subunit.
Authors: Joseph H Davis / Yong Zi Tan / Bridget Carragher / Clinton S Potter / Dmitry Lyumkis / James R Williamson
Abstract: The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as ...The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines.
DateDeposition: Oct 25, 2016 / Header (metadata) release: Nov 16, 2016 / Map release: Dec 14, 2016 / Last update: Feb 14, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8446.png

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Map

Fileemd_8446.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.31 Å/pix.
= 419.2 Å		320 pix
1.31 Å/pix.
= 419.2 Å		320 pix
1.31 Å/pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour Level:0.0262 (by author), 0.0262 (movie #1):
Minimum - Maximum-0.04029261 - 0.12727383
Average (Standard dev.)-0.0011535385 (0.005971469)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin0.00.00.0
Limit319.0319.0319.0
Spacing320320320
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0400.127-0.001

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Supplemental data

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Mask #1

Fileemd_8446_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
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Sample components

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Entire bL17-depleted large ribosomal subunit assembly intermediate - Class D1

EntireName: bL17-depleted large ribosomal subunit assembly intermediate - Class D1
Details: E. coli large ribosome subunit purified from bL17-depleted cells.
Number of components: 1

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Component #1: protein, bL17-depleted large ribosomal subunit assembly intermedi...

ProteinName: bL17-depleted large ribosomal subunit assembly intermediate - Class D1
Details: E. coli large ribosome subunit purified from bL17-depleted cells.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: NCM3722 rplQ::cat, pHSL-rplQ

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: Most of the sucrose was removed by spin concentration
pH: 7.5
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 80 % / Details: 3 microliter of the sample was added..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: In order to account for highly preferred orientation of the specimen, data was 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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 34.27 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 38167.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 3500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2479 / Sampling size: 5 microns
Raw dataEMPIAR-10076 (Title: CryoEM Dataset of L17-Depleted 50S Ribosomal Intermediates
Data size: 50.3
Data #1: Particle stack of L17-depleted 50S Ribosomal Assembly Intermediates [picked particles - multiframe - unprocessed])

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 7345
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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