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- EMDB-1976: Functional reconstitution of the 13-subunit human translation ini... -

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Basic information

Entry
Database: EMDB / ID: EMD-1976
TitleFunctional reconstitution of the 13-subunit human translation initiation factor eIF3.
Map data3d reconstruction of 12mer human translation initiation factor eIF3
Sample
  • Sample: 12mer of the human eukaryotic translation initiation factor eIF3
  • Organelle or cellular component: eIF3 a
  • Organelle or cellular component: eIF3 b
  • Organelle or cellular component: eIF3 c
  • Organelle or cellular component: eIF3 d
  • Organelle or cellular component: eIF3 e
  • Organelle or cellular component: eIF3 f
  • Organelle or cellular component: eIF3 g
  • Organelle or cellular component: eIF3 h
  • Organelle or cellular component: eIF3 i
  • Organelle or cellular component: eIF3 k
  • Organelle or cellular component: eIF3 l
  • Organelle or cellular component: eIF3 m
Keywordstranslation initiation / eukaryotic initiation factor 3 / ribosome / hepatitis C virus / internal ribosome entry site / proteasome / COP9 signalosome
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 29.3 Å
AuthorsQuerol-Audi J / Sun C / Todorovic A / Bai Y / Villa N / Snyder M / Ashchyan J / Lewis C / Hartland A / Gradia S ...Querol-Audi J / Sun C / Todorovic A / Bai Y / Villa N / Snyder M / Ashchyan J / Lewis C / Hartland A / Gradia S / Fraser CS / Doudna JA / Nogales E / Cate JHD
CitationJournal: Proc Natl Acad Sci U S A / Year: 2011
Title: Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).
Authors: Chaomin Sun / Aleksandar Todorovic / Jordi Querol-Audí / Yun Bai / Nancy Villa / Monica Snyder / John Ashchyan / Christopher S Lewis / Abbey Hartland / Scott Gradia / Christopher S Fraser / ...Authors: Chaomin Sun / Aleksandar Todorovic / Jordi Querol-Audí / Yun Bai / Nancy Villa / Monica Snyder / John Ashchyan / Christopher S Lewis / Abbey Hartland / Scott Gradia / Christopher S Fraser / Jennifer A Doudna / Eva Nogales / Jamie H D Cate /
Abstract: Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). ...Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.
History
DepositionOct 8, 2011-
Header (metadata) releaseNov 3, 2011-
Map releaseDec 21, 2011-
UpdateDec 21, 2011-
Current statusDec 21, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.8
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

12mer_emd197...

Downloads & links

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Map

FileDownload / File: emd_1976.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3d reconstruction of 12mer human translation initiation factor eIF3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.36 Å/pix.
x 96 pix.
= 418.56 Å		4.36 Å/pix.
x 96 pix.
= 418.56 Å		4.36 Å/pix.
x 96 pix.
= 418.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.8 / Movie #1: 4.8
Minimum - Maximum-5.17689 - 15.9619
Average (Standard dev.)0.00000000155009 (±0.999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 418.56 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.364.364.36
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z418.560418.560418.560
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/NS969696
D min/max/mean-5.17715.9620.000

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Supplemental data

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Sample components

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Entire : 12mer of the human eukaryotic translation initiation factor eIF3

EntireName: 12mer of the human eukaryotic translation initiation factor eIF3
Components
  • Sample: 12mer of the human eukaryotic translation initiation factor eIF3
  • Organelle or cellular component: eIF3 a
  • Organelle or cellular component: eIF3 b
  • Organelle or cellular component: eIF3 c
  • Organelle or cellular component: eIF3 d
  • Organelle or cellular component: eIF3 e
  • Organelle or cellular component: eIF3 f
  • Organelle or cellular component: eIF3 g
  • Organelle or cellular component: eIF3 h
  • Organelle or cellular component: eIF3 i
  • Organelle or cellular component: eIF3 k
  • Organelle or cellular component: eIF3 l
  • Organelle or cellular component: eIF3 m

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Supramolecule #1000: 12mer of the human eukaryotic translation initiation factor eIF3

SupramoleculeName: 12mer of the human eukaryotic translation initiation factor eIF3
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: Subcomplex containing 12 subunits, missing j subunit
Number unique components: 1
Molecular weightTheoretical: 700 KDa

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Supramolecule #1: eIF3 a

SupramoleculeName: eIF3 a / type: organelle_or_cellular_component / ID: 1 / Name.synonym: eIF3 a / Details: truncated version, contains residues 5-654 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #2: eIF3 b

SupramoleculeName: eIF3 b / type: organelle_or_cellular_component / ID: 2 / Name.synonym: eIF3 b / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: eIF3 c

SupramoleculeName: eIF3 c / type: organelle_or_cellular_component / ID: 3 / Name.synonym: eIF3 c / Details: truncated version, contains residues 302-913 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: eIF3 d

SupramoleculeName: eIF3 d / type: organelle_or_cellular_component / ID: 4 / Name.synonym: eIF3 d / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #5: eIF3 e

SupramoleculeName: eIF3 e / type: organelle_or_cellular_component / ID: 5 / Name.synonym: eIF3 e / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #6: eIF3 f

SupramoleculeName: eIF3 f / type: organelle_or_cellular_component / ID: 6 / Name.synonym: eIF3 f / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #7: eIF3 g

SupramoleculeName: eIF3 g / type: organelle_or_cellular_component / ID: 7 / Name.synonym: eIF3 g / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #8: eIF3 h

SupramoleculeName: eIF3 h / type: organelle_or_cellular_component / ID: 8 / Name.synonym: eIF3 h / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #9: eIF3 i

SupramoleculeName: eIF3 i / type: organelle_or_cellular_component / ID: 9 / Name.synonym: eIF3 i / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #10: eIF3 k

SupramoleculeName: eIF3 k / type: organelle_or_cellular_component / ID: 10 / Name.synonym: eIF3 k / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #11: eIF3 l

SupramoleculeName: eIF3 l / type: organelle_or_cellular_component / ID: 11 / Name.synonym: eIF3 l / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #12: eIF3 m

SupramoleculeName: eIF3 m / type: organelle_or_cellular_component / ID: 12 / Name.synonym: eIF3 m / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: Cytoplasm
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.02 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES, 120 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, 3% trehalose
StainingType: NEGATIVE
Details: Grids with adsorbed protein stained with 3% w/v uranyl acetate for 40 seconds.
GridDetails: 200 mesh Cu grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 340 / Average electron dose: 20 e/Å2 / Details: Data collected using TVIPS camera
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 49000
Sample stageSpecimen holder: Eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsThe particles were selected using an automatic selection program.
CTF correctionDetails: Whole image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 29.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 12457
Final angle assignmentDetails: EMAN2 with initial and final angular steps of 25 and 10 degrees
Final two d classificationNumber classes: 400

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