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- EMDB-0484: Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit -

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Basic information

Entry
Database: EMDB / ID: EMD-0484
TitleRole of Era in Assembly and Homeostasis of the Ribosomal Small Subunit
Map data
SampleEra+YjeQ-treated mature 30S subunit
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsOrtega J / Davis JH / Hao Y / Jahagirdar D / Thurlow B / Basu K / Jain N / Gomez-Blanco J / Britton RA / Vargas J / Guarne A / Woodson SA / Williamson JR / Ortega J
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Role of Era in assembly and homeostasis of the ribosomal small subunit.
Authors: Aida Razi / Joseph H Davis / Yumeng Hao / Dushyant Jahagirdar / Brett Thurlow / Kaustuv Basu / Nikhil Jain / Josue Gomez-Blanco / Robert A Britton / Javier Vargas / Alba Guarné / Sarah A Woodson / James R Williamson / Joaquin Ortega /
Abstract: Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is ...Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.
DateDeposition: Jan 20, 2019 / Header (metadata) release: Jan 30, 2019 / Map release: Jun 26, 2019 / Update: Jul 17, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0901
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0901
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0484.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 304 pix.
= 326.192 Å
1.07 Å/pix.
x 304 pix.
= 326.192 Å
1.07 Å/pix.
x 304 pix.
= 326.192 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 0.0901 / Movie #1: 0.0901
Minimum - Maximum-0.4120274 - 0.6444244
Average (Standard dev.)0.0016175517 (±0.01751076)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 326.192 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z326.192326.192326.192
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS304304304
D min/max/mean-0.4120.6440.002

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Supplemental data

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Sample components

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Entire Era+YjeQ-treated mature 30S subunit

EntireName: Era+YjeQ-treated mature 30S subunit
Details: Mature 30S subunits exposed to Era and YjeQ proteins
Number of components: 1

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Component #1: protein, Era+YjeQ-treated mature 30S subunit

ProteinName: Era+YjeQ-treated mature 30S subunit
Details: Mature 30S subunits exposed to Era and YjeQ proteins
Recombinant expression: No
MassTheoretical: 900 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.5
Support film5 mA
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 298.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 28 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1250.0 - 2750.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 309949
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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