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- EMDB-1347: The eukaryotic translation initiation factors eIF1 and eIF1A indu... -

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Basic information

Entry
Database: EMDB / ID: EMD-1347
TitleThe eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome.
Map dataYeast 40S small ribosomal subunit bound to the translation initiation factors eIF1 and eIF1A
Sample
  • Sample: S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A
  • Complex: 40S
  • Protein or peptide: eIF1
  • Protein or peptide: eIF1AEIF1AX
Function / homologySUI1 domain / Translation initiation factor 1A (eIF-1A) / translation initiation factor activity
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 22.0 Å
AuthorsPassmore LA / Schmeing TM / Maag D / Applefield DJ / Acker MG / Algire MA / Lorsch JR / Ramakrishnan V
CitationJournal: Mol Cell / Year: 2007
Title: The eukaryotic translation initiation factors eIF1 and eIF1A induce an open conformation of the 40S ribosome.
Authors: Lori A Passmore / T Martin Schmeing / David Maag / Drew J Applefield / Michael G Acker / Mikkel A Algire / Jon R Lorsch / V Ramakrishnan /
Abstract: Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two ...Initiation of translation is the process by which initiator tRNA and the start codon of mRNA are positioned in the ribosomal P site. In eukaryotes, one of the first steps involves the binding of two small factors, eIF1 and eIF1A, to the small (40S) ribosomal subunit. This facilitates tRNA binding, allows scanning of mRNA, and maintains fidelity of start codon recognition. Using cryo-EM, we have obtained 3D reconstructions of 40S bound to both eIF1 and eIF1A, and with each factor alone. These structures reveal that together, eIF1 and eIF1A stabilize a conformational change that opens the mRNA binding channel. Biochemical data reveal that both factors accelerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabilizes this interaction. Our results suggest that eIF1 and eIF1A promote an open, scanning-competent preinitiation complex that closes upon start codon recognition and eIF1 release to stabilize ternary complex binding and clamp down on mRNA.
History
DepositionMar 28, 2007-
Header (metadata) releaseMar 28, 2007-
Map releaseJun 6, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.143158
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.143158
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1347.gif

Downloads & links

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Map

FileDownload / File: emd_1347.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast 40S small ribosomal subunit bound to the translation initiation factors eIF1 and eIF1A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.4 Å/pix.
x 192 pix.
= 460.8 Å		2.4 Å/pix.
x 192 pix.
= 460.8 Å		2.4 Å/pix.
x 192 pix.
= 460.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.4 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 2.0 / Movie #1: 3.143158
Minimum - Maximum-11.9367 - 15.5124
Average (Standard dev.)0.000000000438567 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 460.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.42.42.4
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-11.93715.5120.000

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Supplemental data

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Sample components

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Entire : S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A

EntireName: S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A
Components
  • Sample: S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A
  • Complex: 40S
  • Protein or peptide: eIF1
  • Protein or peptide: eIF1AEIF1AX

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Supramolecule #1000: S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A

SupramoleculeName: S. cerevisiae 40S ribosomal subunit bound to eIF1 and eIF1A
type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 3
Molecular weightTheoretical: 1.2 MDa

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Supramolecule #1: 40S

SupramoleculeName: 40S / type: complex / ID: 1 / Name.synonym: small ribosomal subunit / Details: Purified from Saccharomyces cerevisiae. / Ribosome-details: ribosome-eukaryote: SSU 40S
Molecular weightExperimental: 1.2 MDa

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Macromolecule #1: eIF1

MacromoleculeName: eIF1 / type: protein_or_peptide / ID: 1 / Name.synonym: SUI1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 12 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pTYB2
SequenceGO: translation initiation factor activity / InterPro: SUI1 domain

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Macromolecule #2: eIF1A

MacromoleculeName: eIF1A / type: protein_or_peptide / ID: 2 / Name.synonym: TIF11 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 17 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pTYB2
SequenceGO: translation initiation factor activity / InterPro: Translation initiation factor 1A (eIF-1A)

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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

BufferpH: 7.4
Details: 20 mM HEPES-KOH pH 7.4, 100 mM potassium acetate, 2.5 mM magnesium acetate, 2 mM DTT
GridDetails: Quantifoil R2/2, 200 mesh, Cu/Rh
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: 40S subunits were incubated on ice at a concentration of 500-750 nM with a five-fold molar excess of each of eIF1 and eIF1A. Samples were diluted to a final concentration of 50 nM or 75 nM ...Method: 40S subunits were incubated on ice at a concentration of 500-750 nM with a five-fold molar excess of each of eIF1 and eIF1A. Samples were diluted to a final concentration of 50 nM or 75 nM immediately before freezing. 4 ul was applied to one side of a glow-discharged grid, blotted on both sides and flash frozen in liquid ethane.

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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 mm / Nominal defocus max: 5.3 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 95 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 200
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 6 µm / Number real images: 82 / Average electron dose: 15 e/Å2
Details: Micrographs were digitized using a KZA scanner (MRC, Cambridge).
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping
Final two d classificationNumber classes: 490
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 31054
DetailsParticles were manually selected using Ximdisp.

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Atomic model buiding 1

Initial modelPDB ID:

1s1h
PDB Unreleased entry

SoftwareName: Situs
DetailsProtocol: rigid body. A model of S. cerevisiae 40S (PDB 1S1H) was manually placed in the density using Chimera and refined as a rigid structure using Situs.
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation

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