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- EMDB-1775: Understanding Ribosome Assembly: the Structure of in vivo Assembl... -

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Basic information

Entry
Database: EMDB / ID: EMD-1775
TitleUnderstanding Ribosome Assembly: the Structure of in vivo Assembled Immature 30S Subunits Revealed by Cryo-electron Microscopy
Map dataSurface rendering of 30S ribosomal subunit from wild type E. coli cells. Map was used as a control.
Sample
  • Sample: Reconstruction of the mature 30S subunit from wild type E.coli cells
  • Complex: Small Ribosomal Subunit
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.1 Å
AuthorsJomaa A / Stewart G / Benito JM / Zielke R / Campbell T / Maddock J / Brown E / Ortega J
CitationJournal: RNA / Year: 2011
Title: Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy.
Authors: Ahmad Jomaa / Geordie Stewart / Jaime Martín-Benito / Ryszard Zielke / Tracey L Campbell / Janine R Maddock / Eric D Brown / Joaquin Ortega /
Abstract: Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure ...Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.
History
DepositionAug 17, 2010-
Header (metadata) releaseSep 3, 2010-
Map releaseApr 8, 2011-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1775.tif

Downloads & links

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Map

FileDownload / File: emd_1775.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSurface rendering of 30S ribosomal subunit from wild type E. coli cells. Map was used as a control.
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.01
Minimum - Maximum-0.0265466 - 0.0791865
Average (Standard dev.)0.000318398 (±0.00456445)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 325.12 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z325.120325.120325.120
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0270.0790.000

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Supplemental data

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Sample components

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Entire : Reconstruction of the mature 30S subunit from wild type E.coli cells

EntireName: Reconstruction of the mature 30S subunit from wild type E.coli cells
Components
  • Sample: Reconstruction of the mature 30S subunit from wild type E.coli cells
  • Complex: Small Ribosomal Subunit

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Supramolecule #1000: Reconstruction of the mature 30S subunit from wild type E.coli cells

SupramoleculeName: Reconstruction of the mature 30S subunit from wild type E.coli cells
type: sample / ID: 1000
Details: The sample was thawed from storage at -80 degrees Celcius before being loaded onto the grid.
Number unique components: 1
Molecular weightTheoretical: 800 KDa

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Supramolecule #1: Small Ribosomal Subunit

SupramoleculeName: Small Ribosomal Subunit / type: complex / ID: 1 / Name.synonym: 30S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 850 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

Concentration5.8 mg/mL
BufferpH: 7.5
Details: 10 mM Tris-HCl pH 7.5, 10 mM magnesium acetate, 60 mM NH4Cl, 3 mM 2-mercaptoethanol
GridDetails: 400 Mesh Copper Grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: OTHER / Details: Vitrification instrument: FEI vitrobot / Method: Blot 7 Seconds Twice

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.0 mm / Nominal defocus max: 3.9 µm / Nominal defocus min: 0.65 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 93 K / Max: 93 K / Average: 93 K
DateDec 10, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 29 / Average electron dose: 10 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: Each Micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp / Number images used: 25803
DetailsParticles were picked with BOXER

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Atomic model buiding 1

Initial modelPDB ID:

2z4k
PDB Unreleased entry

SoftwareName: Situs
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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