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- EMDB-2450: Cryo-EM map of the CSFV IRES in complex with the small ribosomal ... -

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Entry
Database: EMDB / ID: EMD-2450
TitleCryo-EM map of the CSFV IRES in complex with the small ribosomal 40S subunit and DHX29
Map dataReconstruction of a mutant Classical Swine Fever Virus IRES bound to the Rabbit 40S subunit and DHX29.
Sample
  • Sample: CSFV IRES truncated from domain II, in complex with the Rabbit small ribosomal 40S subunit and to DHX29
  • Complex: eukaryotic small ribosmal subunit
  • RNA: Internal Ribosomal Entry Site
  • Protein or peptide: DHX29
KeywordsInternal Ribosomal Entry Site / 5'-end independent initiation / HCV-like IRES
Biological speciesOryctolagus cuniculus (rabbit) / Classical swine fever virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsHashem Y / des Georges A / Dhote A / Langlois R / Liao HY / Grassucci RA / Pestova TV / Hellen CUT / Frank J
CitationJournal: Nature / Year: 2013
Title: Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit.
Authors: Yaser Hashem / Amedee des Georges / Vidya Dhote / Robert Langlois / Hstau Y Liao / Robert A Grassucci / Tatyana V Pestova / Christopher U T Hellen / Joachim Frank /
Abstract: Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of ...Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5-7, 9-12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.
History
DepositionSep 7, 2013-
Header (metadata) releaseSep 18, 2013-
Map releaseNov 13, 2013-
UpdateNov 27, 2013-
Current statusNov 27, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4c4q
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4c4q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2450-40S...

Downloads & links

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Map

FileDownload / File: emd_2450.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a mutant Classical Swine Fever Virus IRES bound to the Rabbit 40S subunit and DHX29.
Voxel sizeX=Y=Z: 2.245 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.09494244 - 0.31092703
Average (Standard dev.)-0.00031056 (±0.02028314)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 493.89996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2452.2452.245
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z493.900493.900493.900
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0950.311-0.000

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Supplemental data

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Sample components

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Entire : CSFV IRES truncated from domain II, in complex with the Rabbit sm...

EntireName: CSFV IRES truncated from domain II, in complex with the Rabbit small ribosomal 40S subunit and to DHX29
Components
  • Sample: CSFV IRES truncated from domain II, in complex with the Rabbit small ribosomal 40S subunit and to DHX29
  • Complex: eukaryotic small ribosmal subunit
  • RNA: Internal Ribosomal Entry Site
  • Protein or peptide: DHX29

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Supramolecule #1000: CSFV IRES truncated from domain II, in complex with the Rabbit sm...

SupramoleculeName: CSFV IRES truncated from domain II, in complex with the Rabbit small ribosomal 40S subunit and to DHX29
type: sample / ID: 1000 / Oligomeric state: one 40S, one CSFV IRES and one DHX29 / Number unique components: 3
Molecular weightTheoretical: 1.75 MDa

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Supramolecule #1: eukaryotic small ribosmal subunit

SupramoleculeName: eukaryotic small ribosmal subunit / type: complex / ID: 1 / Name.synonym: 40S subunit / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S, SSU RNA 18S
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: blood / Cell: reticulocytes
Molecular weightTheoretical: 1.5 MDa

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Macromolecule #1: Internal Ribosomal Entry Site

MacromoleculeName: Internal Ribosomal Entry Site / type: rna / ID: 1 / Name.synonym: IRES / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Classical swine fever virus / synonym: CSFV
Molecular weightTheoretical: 100 KDa
SequenceString: GACUAGCCGU AGUGGCGAGC UCCCUGGGUG GUCUAAGUCC UGAGUACAGG ACAGUCGUCA GUAGUUCGAC GUGAGCACUA GCCCACCUCG AGAUGCUACG UGGACGAGGG CAUGCCCAAG ACACACCUUA ACCCUGGCGG GGGUCGCUAG GGUGAAAUCA CAUUAUGUGA ...String:
GACUAGCCGU AGUGGCGAGC UCCCUGGGUG GUCUAAGUCC UGAGUACAGG ACAGUCGUCA GUAGUUCGAC GUGAGCACUA GCCCACCUCG AGAUGCUACG UGGACGAGGG CAUGCCCAAG ACACACCUUA ACCCUGGCGG GGGUCGCUAG GGUGAAAUCA CAUUAUGUGA UGGGGGUACG ACCUGAUAGG GUGCUGCAGA GGCCCACUAG CAGGCUAGUA UAAAAAUCUC UGC

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Macromolecule #2: DHX29

MacromoleculeName: DHX29 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 150 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.105 mg/mL
BufferpH: 7.5
Details: 20 mM Tris, 75 mM KCl, 5 mM Mg, 2 mM DTT and 0.25 mM spermidine
GridDetails: 300 mesh copper/molybdenum holey carbon-coated Quantifoil 2/4 grid (Quantifoil Micro Tools GmbH) containing an additional continuous thin layer of carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK II
Method: 30 seconds waiting after sample deposition on the grid, blotting for seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51570 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder: Gatan CT3500 side-entry cryo-holder / Specimen holder model: OTHER
TemperatureAverage: 110 K
DateFeb 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 12000 / Average electron dose: 12 e/Å2 / Bits/pixel: 32
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: Spider, Relion / Number images used: 72900
DetailsThe particles constituting this map were sorted from a larger dataset using RELION

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