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- EMDB-1923: Ribosome Assembly Factors Prevent Premature Translation Initiatio... -

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Basic information

Entry
Database: EMDB / ID: EMD-1923
TitleRibosome Assembly Factors Prevent Premature Translation Initiation by 40S Assembly Intermediates
Map dataSurface render Pre-40s Nob1 depletion map
Sample
  • Sample: S. cerevisiae pre-40S ribosomal particle depleted for Nob1
  • Complex: pre-40S
Keywordspre-40S / 40S intermediate / Nob1 depletion
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsStrunk BS / Loucks CR / Su M / Vashisth H / Cheng S / Schilling J / BrooksIII CL / Karbstein K / Skiniotis G
CitationJournal: Science / Year: 2011
Title: Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates.
Authors: Bethany S Strunk / Cherisse R Loucks / Min Su / Harish Vashisth / Shanshan Cheng / Justin Schilling / Charles L Brooks / Katrin Karbstein / Georgios Skiniotis /
Abstract: Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we ...Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.
History
DepositionJul 5, 2011-
Header (metadata) releaseNov 4, 2011-
Map releaseNov 4, 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: 0.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.5
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1923.png

Downloads & links

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Map

FileDownload / File: emd_1923.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSurface render Pre-40s Nob1 depletion map
Voxel sizeX=Y=Z: 2.24 Å
Density
Contour LevelBy AUTHOR: 0.5 / Movie #1: 0.5
Minimum - Maximum-4.08762 - 6.29259
Average (Standard dev.)0.00444547 (±0.582385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 358.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.242.242.24
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-4.0886.2930.004

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Supplemental data

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Sample components

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Entire : S. cerevisiae pre-40S ribosomal particle depleted for Nob1

EntireName: S. cerevisiae pre-40S ribosomal particle depleted for Nob1
Components
  • Sample: S. cerevisiae pre-40S ribosomal particle depleted for Nob1
  • Complex: pre-40S

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Supramolecule #1000: S. cerevisiae pre-40S ribosomal particle depleted for Nob1

SupramoleculeName: S. cerevisiae pre-40S ribosomal particle depleted for Nob1
type: sample / ID: 1000 / Details: The sample was monodisperse / Number unique components: 1
Molecular weightTheoretical: 1.5 MDa

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Supramolecule #1: pre-40S

SupramoleculeName: pre-40S / type: complex / ID: 1 / Name.synonym: pre-40S / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Bakers' yeast
Molecular weightTheoretical: 1.5 MDa

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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

Concentration1.5 mg/mL
BufferpH: 7.5
Details: 50mM Tris-Cl, 100mM NaCl, 10mM MgCl2, 0.075% NP40, 1mM imidazole, 2mM EGTA, 10 mM BME.
GridDetails: quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 85 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 66964 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: OTHER
TemperatureMin: 89 K / Max: 89 K / Average: 89 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 135,000 times magnification
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 2.24 µm / Number real images: 310 / Average electron dose: 16 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final angle assignmentDetails: EMAN convention
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Details: Final map was filtered to 20A resolution. / Number images used: 9132
DetailsManual particle selection

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Atomic model buiding 1

Initial modelPDB ID:

3o2z
PDB Unreleased entry

SoftwareName: NAMD
DetailsProtocol: MDFF. Manual docking of modified 3o2z model followed by MDFF. See publication for further details.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: CC

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