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- EMDB-2670: Occupied class from the supervised classification of a negatively... -

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Entry
Database: EMDB / ID: EMD-2670
TitleOccupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
Map dataOccupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
Sample
  • Sample: Occupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
  • Complex: 40S
  • Protein or peptide: eIF3Eukaryotic initiation factor 3
  • Protein or peptide: eIF1
KeywordsEukaryotic translation / Translation initiation / eIF3 / 40S / Ribosome
Biological speciesLachancea kluyveri (fungus)
Methodsingle particle reconstruction / negative staining / Resolution: 28.1 Å
AuthorsAylett CHS / Boehringer D / Erzberger JP / Zhang S / Schaefer T / Ban N
CitationJournal: Cell / Year: 2014
Title: Molecular architecture of the 40S⋅eIF1⋅eIF3 translation initiation complex.
Authors: Jan P Erzberger / Florian Stengel / Riccardo Pellarin / Suyang Zhang / Tanja Schaefer / Christopher H S Aylett / Peter Cimermančič / Daniel Boehringer / Andrej Sali / Ruedi Aebersold / Nenad Ban /
Abstract: Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, ...Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.
History
DepositionJun 4, 2014-
Header (metadata) releaseJun 25, 2014-
Map releaseSep 10, 2014-
UpdateSep 10, 2014-
Current statusSep 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.75
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_2670.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOccupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
Voxel sizeX=Y=Z: 5.46 Å
Density
Contour LevelBy AUTHOR: 0.75 / Movie #1: 0.75
Minimum - Maximum-2.68660688 - 7.31199741
Average (Standard dev.)0.07138462 (±0.63173282)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 393.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.465.465.46
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z393.120393.120393.120
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-2.6877.3120.071

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Supplemental data

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Sample components

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Entire : Occupied class from the supervised classification of a negatively...

EntireName: Occupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
Components
  • Sample: Occupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
  • Complex: 40S
  • Protein or peptide: eIF3Eukaryotic initiation factor 3
  • Protein or peptide: eIF1

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Supramolecule #1000: Occupied class from the supervised classification of a negatively...

SupramoleculeName: Occupied class from the supervised classification of a negatively stained Lachancea kluyveri 40S-eIF1-eIF3 complex.
type: sample / ID: 1000
Details: This is the occupied class from two-way supervised classification of a single data set. Density corresponding to eIF3 is visible in this map only. The unoccupied map is provided for the purposes of comparison.
Number unique components: 3

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Supramolecule #1: 40S

SupramoleculeName: 40S / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Lachancea kluyveri (fungus)
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: eIF3

MacromoleculeName: eIF3 / type: protein_or_peptide / ID: 1 / Details: Includes eIF3 polypeptides: A, B, C, I, G, J / Recombinant expression: Yes
Source (natural)Organism: Lachancea kluyveri (fungus)
Molecular weightTheoretical: 400 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pLIC-HMK

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Macromolecule #2: eIF1

MacromoleculeName: eIF1 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Lachancea kluyveri (fungus)
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pLIC-HMK

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.6
Details: 750 mM sucrose, 75 mM KCl, 25 mM Hepes pH 7.6, 10 mM MgCl2, 2 mM TCEP and 5 uM glutaraldehyde.
StainingType: NEGATIVE
Details: A fine film of carbon produced upon a cleaved mica substrate was applied first to the surface of the isolated sample and then to a 50 mM solution of uranyl acetate. The film was recovered ...Details: A fine film of carbon produced upon a cleaved mica substrate was applied first to the surface of the isolated sample and then to a 50 mM solution of uranyl acetate. The film was recovered onto the surface of holey carbon copper grids (Quantifoil) and excess stain removed by blotting with filter paper.
GridDetails: Quantifoil R1/2
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 83000
Sample stageSpecimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 135,000 times magnification
DateOct 22, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per frame
Final angle assignmentDetails: SPIDER VO EA: 6 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.1 Å / Resolution method: OTHER / Software - Name: Imagic-5, SPIDER / Number images used: 12611
DetailsImagic 5 and SPIDER

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