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- PDB-4c4q: Cryo-EM map of the CSFV IRES in complex with the small ribosomal ... -

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Basic information

Entry
Database: PDB / ID: 4c4q
TitleCryo-EM map of the CSFV IRES in complex with the small ribosomal 40S subunit and DHX29
ComponentsINTERNAL RIBOSOMAL ENTRY SITE
KeywordsRNA / INTERNAL RIBOSOMAL ENTRY SITE / 5'-END INDEPENDENT INITIATION / HCV-LIKE IRES
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesCLASSICAL SWINE FEVER VIRUS
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsHashem, Y. / desGeorges, A. / Dhote, V. / Langlois, R. / Liao, H.Y. / Grassucci, R.A. / Pestova, T.V. / Hellen, C.U.T. / 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, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 30, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2013Group: Database references
Revision 1.2Dec 4, 2013Group: Database references
Revision 2.0Aug 2, 2017Group: Atomic model / Data collection / Derived calculations
Category: atom_site / em_software / struct_conn
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _em_software.image_processing_id / _em_software.name

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Structure visualization

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Assembly

Deposited unit
N: INTERNAL RIBOSOMAL ENTRY SITE


Theoretical massNumber of molelcules
Total (without water)75,3521
Polymers75,3521
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain INTERNAL RIBOSOMAL ENTRY SITE /


Mass: 75351.680 Da / Num. of mol.: 1 / Fragment: DOMAIN III OF CSFV IRES, RESIDUES 128-360 / Source method: obtained synthetically
Details: AS DOMAIN II IS TRUNCATED, THE IRES STRUCTURED BODY CONSISTS MAINLY ON DOMAINS III (RESIDUES 128-360)
Source: (synth.) CLASSICAL SWINE FEVER VIRUS / References: GenBank: J04358

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CSFV IRES TRUNCATED FROM DOMAIN II, IN COMPLEX WITH THE RABBIT SMALL RIBOSOMAL 40S SUBUNIT AND TO DHX29
Type: RIBOSOME
Buffer solutionName: 20 MM TRIS, 75 MM KCL, 5MM MG, 2 MM DTT AND 0.25 MM SPERMIDINE
pH: 7.5
Details: 20 MM TRIS, 75 MM KCL, 5MM MG, 2 MM DTT AND 0.25 MM SPERMIDINE
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: CARBON
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 120, 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 imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Feb 1, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 51570 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.26 mm
Specimen holderTemperature: 110 K
Image recordingElectron dose: 12 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 2000
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1RELION3D reconstruction
2SPIDER3D reconstruction
CTF correctionDetails: EACH PARTICLE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: TEMPLATE MATCHING / Resolution: 8.5 Å / Num. of particles: 72900 / Actual pixel size: 2.245 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2450. (DEPOSITION ID: 11933)
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Details: REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 2XZM

2xzm
PDB Unreleased entry

RefinementHighest resolution: 8.5 Å
Refinement stepCycle: LAST / Highest resolution: 8.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms0 4986 0 0 4986

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