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- EMDB-2199: Architecture of human translation initiation factor 3 -

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Basic information

Entry
Database: EMDB / ID: EMD-2199
TitleArchitecture of human translation initiation factor 3
Map datareconstruction using maximum-likelihood
Sample
  • Sample: PCI MPN core of the human eukaryotic translation initiation factor eIF3 bound to the IIIabc domain of Hepatitis C IRES
  • Protein or peptide: eIF3 a
  • Protein or peptide: eIF3 c
  • Protein or peptide: eIF3 e
  • Protein or peptide: eIF3 f
  • Protein or peptide: eIF3 h
  • Protein or peptide: eIF3 k
  • Protein or peptide: eIF3 l
  • Protein or peptide: eIF3 m
  • RNA: IIIabc
Keywordstranslation initiation / eukaryotic initiation factor 3 / ribosome / proteasome / PCI-MPN domains
Biological speciesHomo sapiens (human) / Hepatitis C virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsQuerol-Audi J / Sun C / Mortimer S / Arias-Palomo E / Vogan J / Doudna J / Nogales E / Cate J
CitationJournal: Nucleic Acids Res / Year: 2013
Title: Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.
Authors: Chaomin Sun / Jordi Querol-Audí / Stefanie A Mortimer / Ernesto Arias-Palomo / Jennifer A Doudna / Eva Nogales / Jamie H D Cate /
Abstract: The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) ...The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.
History
DepositionSep 12, 2012-
Header (metadata) releaseSep 19, 2012-
Map releaseMay 8, 2013-
UpdateSep 4, 2013-
Current statusSep 4, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2199_emd_219...

Downloads & links

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Map

FileDownload / File: emd_2199.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction using maximum-likelihood
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.67 Å/pix.
x 72 pix.
= 336.24 Å		4.67 Å/pix.
x 72 pix.
= 336.24 Å		4.67 Å/pix.
x 72 pix.
= 336.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.67 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.06941439 - 0.12644935
Average (Standard dev.)0.0003444 (±0.00859947)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 336.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.674.674.67
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z336.240336.240336.240
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-0.0690.1260.000

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Supplemental data

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Sample components

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Entire : PCI MPN core of the human eukaryotic translation initiation facto...

EntireName: PCI MPN core of the human eukaryotic translation initiation factor eIF3 bound to the IIIabc domain of Hepatitis C IRES
Components
  • Sample: PCI MPN core of the human eukaryotic translation initiation factor eIF3 bound to the IIIabc domain of Hepatitis C IRES
  • Protein or peptide: eIF3 a
  • Protein or peptide: eIF3 c
  • Protein or peptide: eIF3 e
  • Protein or peptide: eIF3 f
  • Protein or peptide: eIF3 h
  • Protein or peptide: eIF3 k
  • Protein or peptide: eIF3 l
  • Protein or peptide: eIF3 m
  • RNA: IIIabc

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Supramolecule #1000: PCI MPN core of the human eukaryotic translation initiation facto...

SupramoleculeName: PCI MPN core of the human eukaryotic translation initiation factor eIF3 bound to the IIIabc domain of Hepatitis C IRES
type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: Subcomplex containing 8 core subunits plus IIIabc
Number unique components: 9
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: eIF3 a

MacromoleculeName: eIF3 a / type: protein_or_peptide / ID: 1 / Details: truncated version, contains residues 5-654 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: eIF3 c

MacromoleculeName: eIF3 c / type: protein_or_peptide / ID: 2 / Details: truncated version, contains residues 302-913 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: eIF3 e

MacromoleculeName: eIF3 e / type: protein_or_peptide / ID: 3 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #4: eIF3 f

MacromoleculeName: eIF3 f / type: protein_or_peptide / ID: 4 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #5: eIF3 h

MacromoleculeName: eIF3 h / type: protein_or_peptide / ID: 5 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #6: eIF3 k

MacromoleculeName: eIF3 k / type: protein_or_peptide / ID: 6 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #7: eIF3 l

MacromoleculeName: eIF3 l / type: protein_or_peptide / ID: 7 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #8: eIF3 m

MacromoleculeName: eIF3 m / type: protein_or_peptide / ID: 8 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #9: IIIabc

MacromoleculeName: IIIabc / type: rna / ID: 9 / Classification: OTHER / Structure: OTHER / Synthetic?: Yes
Source (natural)Organism: Hepatitis C virus

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
Details: 20mM Hepes, 75mM KCl, 0.5mM EDTA, 1mM DTT, 2mM MgCl2, 3% trehalose
StainingType: NEGATIVE / Details: vitrified
GridDetails: C-flat plasma cleaned with Solarus
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TECNAI F20
DateAug 11, 2011
Image recordingAverage electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailsautomatic particle selection with template correlator
CTF correctionDetails: each image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Software - Name: ML3D / Number images used: 15921

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