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- EMDB-2764: CryoEM structure of the 40S-eIF1-eIF1A preinitiation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2764
TitleCryoEM structure of the 40S-eIF1-eIF1A preinitiation complex
Map dataReconstruction of 40S-eIF1-eIF1A preinitiation complex
Sample
  • Sample: 40S-eIF1-eIF1A preinitiation complex
  • Complex: Ribosome small subunit
  • Protein or peptide: Eukaryotic initiation factor 1
  • Protein or peptide: Eukaryotic initiation factor 1A
KeywordsEukaryotic translation initiation / 48S / small ribosome subunit.
Function / homology
Function and homology information


formation of translation initiation ternary complex / translation reinitiation / formation of cytoplasmic translation initiation complex / multi-eIF complex / eukaryotic 43S preinitiation complex / formation of translation preinitiation complex / eukaryotic 48S preinitiation complex / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition ...formation of translation initiation ternary complex / translation reinitiation / formation of cytoplasmic translation initiation complex / multi-eIF complex / eukaryotic 43S preinitiation complex / formation of translation preinitiation complex / eukaryotic 48S preinitiation complex / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / Formation of a pool of free 40S subunits / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal small subunit binding / 90S preribosome / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / translation regulator activity / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translation initiation factor binding / translation initiation factor activity / rescue of stalled ribosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / protein kinase C binding / maintenance of translational fidelity / cytoplasmic stress granule / modification-dependent protein catabolic process / protein tag activity / rRNA processing / ribosomal small subunit biogenesis / double-stranded RNA binding / small ribosomal subunit rRNA binding / ribosome binding / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / translation / ribonucleoprotein complex / positive regulation of protein phosphorylation / mRNA binding / ubiquitin protein ligase binding / nucleolus / protein kinase binding / RNA binding / zinc ion binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic translation initiation factor SUI1 / SUI1 domain superfamily / Translation initiation factor SUI1 / Translation initiation factor SUI1 family profile. / SUI1 domain / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / RNA-binding domain, S1, IF1 type ...Eukaryotic translation initiation factor SUI1 / SUI1 domain superfamily / Translation initiation factor SUI1 / Translation initiation factor SUI1 family profile. / SUI1 domain / Translation initiation factor 1A (eIF-1A), conserved site / Eukaryotic initiation factor 1A signature. / eukaryotic translation initiation factor 1A / Translation initiation factor 1A (eIF-1A) / RNA-binding domain, S1, IF1 type / Translation initiation factor 1A / IF-1 / S1 domain IF1 type profile. / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / : / Ribosomal protein S12e signature. / Ribosomal protein S12e / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / Small (40S) ribosomal subunit Asc1/RACK1 / S27a-like superfamily / 40S Ribosomal protein S10 / Ribosomal protein S10, eukaryotic/archaeal / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S2, eukaryotic/archaeal / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / : / Ribosomal protein S30 / Ribosomal protein S30 / Ribosomal protein S7e signature. / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein S3Ae, conserved site / Ribosomal protein S3Ae signature. / Ribosomal protein S19e / Ribosomal protein S19e / Ribosomal_S19e / Ribosomal protein S27e signature. / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / 40S ribosomal protein S4, C-terminal domain / 40S ribosomal protein S4 C-terminus / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. / Ribosomal protein S19A/S15e / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / Ribosomal protein S6, eukaryotic / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / : / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S4e, N-terminal / RS4NT (NUC023) domain / 40S ribosomal protein S11, N-terminal / Ribosomal_S17 N-terminal / Ribosomal protein S7e / Ribosomal protein S7e / Ribosomal protein S4, KOW domain / Ribosomal protein S4e / Ribosomal protein S4e, central region / Ribosomal protein S4e, central domain superfamily / Ribosomal family S4e / Ribosomal protein S6/S6e/A/B/2, conserved site / Ribosomal protein S6e signature. / Ribosomal protein S23, eukaryotic/archaeal / Ribosomal protein S24e / Ribosomal protein S24e / Ribosomal protein S27 / Ribosomal protein S27, zinc-binding domain superfamily / Ribosomal protein S27 / Ribosomal protein S8e / Ribosomal protein S17, archaeal/eukaryotic / Ribosomal protein S28e conserved site / Ribosomal protein S28e signature. / Ribosomal protein S3Ae / Ribosomal S3Ae family / Ribosomal S3Ae family / Ribosomal protein S28e
Similarity search - Domain/homology
KLLA0B11231p / Small ribosomal subunit protein eS32A / Small ribosomal subunit protein uS11 / Eukaryotic translation initiation factor eIF-1 / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 1A / Ubiquitin-ribosomal protein eS31 fusion protein / KLLA0F25542p / KLLA0F18040p / KLLA0F09812p ...KLLA0B11231p / Small ribosomal subunit protein eS32A / Small ribosomal subunit protein uS11 / Eukaryotic translation initiation factor eIF-1 / Small ribosomal subunit protein eS28 / Eukaryotic translation initiation factor 1A / Ubiquitin-ribosomal protein eS31 fusion protein / KLLA0F25542p / KLLA0F18040p / KLLA0F09812p / KLLA0F07843p / 40S ribosomal protein S12 / Small ribosomal subunit protein eS6 / KLLA0E23673p / 40S ribosomal protein S8 / Small ribosomal subunit protein uS2 / KLLA0E12277p / 40S ribosomal protein S27 / Small ribosomal subunit protein uS14 / KLLA0D10659p / 40S ribosomal protein S3 / 40S ribosomal protein S26 / 40S ribosomal protein S7 / 40S ribosomal protein S24 / 40S ribosomal protein S30 / KLLA0B08173p / Small ribosomal subunit protein uS8 / 40S ribosomal protein S25 / Small ribosomal subunit protein eS1 / 40S ribosomal protein S4 / KLLA0B01562p / KLLA0B01474p / KLLA0A10483p / KLLA0A07194p / Small ribosomal subunit protein eS21 / Small ribosomal subunit protein uS9
Similarity search - Component
Biological speciesKluyveromyces lactis (yeast) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsHussain T / Llacer JL / Fernandez IS / Savva CG / Ramakrishnan V
CitationJournal: Cell / Year: 2014
Title: Structural changes enable start codon recognition by the eukaryotic translation initiation complex.
Authors: Tanweer Hussain / Jose L Llácer / Israel S Fernández / Antonio Munoz / Pilar Martin-Marcos / Christos G Savva / Jon R Lorsch / Alan G Hinnebusch / V Ramakrishnan /
Abstract: During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the ...During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.
History
DepositionAug 26, 2014-
Header (metadata) releaseOct 1, 2014-
Map releaseNov 5, 2014-
UpdateDec 3, 2014-
Current statusDec 3, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j80
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2764.png

emd_2764.png

Downloads & links

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Map

FileDownload / File: emd_2764.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 40S-eIF1-eIF1A preinitiation complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å		1.34 Å/pix.
x 300 pix.
= 402. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.065 / Movie #1: 0.065
Minimum - Maximum-0.22107875 - 0.6015414
Average (Standard dev.)0.00040093 (±0.02360408)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 402.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z402.000402.000402.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2210.6020.000

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Supplemental data

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Supplemental map: emd 2764 half map 1.map

Fileemd_2764_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: emd 2764 half map 2.map

Fileemd_2764_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 40S-eIF1-eIF1A preinitiation complex

EntireName: 40S-eIF1-eIF1A preinitiation complex
Components
  • Sample: 40S-eIF1-eIF1A preinitiation complex
  • Complex: Ribosome small subunit
  • Protein or peptide: Eukaryotic initiation factor 1
  • Protein or peptide: Eukaryotic initiation factor 1A

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Supramolecule #1000: 40S-eIF1-eIF1A preinitiation complex

SupramoleculeName: 40S-eIF1-eIF1A preinitiation complex / type: sample / ID: 1000 / Oligomeric state: 1 / Number unique components: 3
Molecular weightTheoretical: 1.23 MDa

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Supramolecule #1: Ribosome small subunit

SupramoleculeName: Ribosome small subunit / type: complex / ID: 1 / Name.synonym: 40S / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S, SSU RNA 18S
Source (natural)Organism: Kluyveromyces lactis (yeast)
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #1: Eukaryotic initiation factor 1

MacromoleculeName: Eukaryotic initiation factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: eIF1 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 12.3 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pTYB2
SequenceUniProtKB: Eukaryotic translation initiation factor eIF-1

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Macromolecule #2: Eukaryotic initiation factor 1A

MacromoleculeName: Eukaryotic initiation factor 1A / type: protein_or_peptide / ID: 2 / Name.synonym: eIF1A / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightTheoretical: 17.4 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pTYB2
SequenceUniProtKB: Eukaryotic translation initiation factor 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

Concentration0.17 mg/mL
BufferpH: 6.5
Details: 20mM MES-KOH, 40mM K-acetate, 10mM NH4-acetate, 8mM Mg-acetate, 2mM DTT
GridDetails: Quantifoil R2/2 400 mesh copper grids with 4-5 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I / Timed resolved state: 30 second incubation time / Method: Blot for 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 78,000 times magnification
DetailsComplete dataset was collected in 5 non-consecutive days
DateOct 28, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1791 / Average electron dose: 28 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 0.004 µm / Nominal defocus min: 0.0016 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsParticles were semi-automatically picked with the e2boxer.py program of EMAN2
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 100709
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry


Chain - Chain ID: 2
SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3j80:
CryoEM structure of 40S-eIF1-eIF1A preinitiation complex

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Atomic model buiding 2

Initial modelPDB ID:

3u5c
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor, FSC
Output model

PDB-3j80:
CryoEM structure of 40S-eIF1-eIF1A preinitiation complex

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