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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 dataEra YjeQ-treated_30S
Sample
  • Complex: Era+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 ...Ortega 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
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health ResearchPJT-153044 Canada
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 ...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.
History
DepositionJan 20, 2019-
Header (metadata) releaseJan 30, 2019-
Map releaseJun 26, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: RCSB / Status: Released

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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

emd_0484.png

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)
AnnotationEra YjeQ-treated_30S
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
Components
  • Complex: Era+YjeQ-treated mature 30S subunit

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Supramolecule #1: Era+YjeQ-treated mature 30S subunit

SupramoleculeName: Era+YjeQ-treated mature 30S subunit / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#16
Details: Mature 30S subunits exposed to Era and YjeQ proteins
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12
Molecular weightTheoretical: 900 KDa

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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
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 28.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 764781
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2avy
PDB Unreleased entry

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1.0.)
Final 3D classificationSoftware - Name: RELION (ver. 2.1.0.)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1.0.)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1.0.) / Number images used: 309949
FSC plot (resolution estimation)

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