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- EMDB-1138: Structure of the hepatitis C virus IRES bound to the human 80S ri... -

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Basic information

Entry
Database: EMDB / ID: EMD-1138
TitleStructure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES.
Map dataCryo-EM map of the hepatitis C virus internal ribosome entry site in complex with human 80S ribosome
Sample
  • Sample: IRES-80S ribosome complex
  • Organelle or cellular component: ribosome
  • RNA: HCV IRESHepatitis C virus internal ribosome entry site
Biological speciesHomo sapiens (human) / Hepatitis C virus
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 15.0 Å
AuthorsBoehringer D / Thermann R / Ostareck-Lederer A / Lewis JD / Stark H
CitationJournal: Structure / Year: 2005
Title: Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES.
Authors: Daniel Boehringer / Rolf Thermann / Antje Ostareck-Lederer / Joe D Lewis / Holger Stark /
Abstract: Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. ...Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.
History
DepositionJul 27, 2005-
Header (metadata) releaseJul 27, 2005-
Map releaseJul 27, 2006-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing 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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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1138.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the hepatitis C virus internal ribosome entry site in complex with human 80S ribosome
Voxel sizeX=Y=Z: 3.6 Å
Density
Contour Level1: 0.0521 / Movie #1: 0.065
Minimum - Maximum-0.423707 - 0.541096
Average (Standard dev.)-0.000000000004334 (±0.0509359)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 460.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z460.800460.800460.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-90-90-190
NX/NY/NZ180180380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.4240.541-0.000

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Supplemental data

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Sample components

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Entire : IRES-80S ribosome complex

EntireName: IRES-80S ribosome complex
Components
  • Sample: IRES-80S ribosome complex
  • Organelle or cellular component: ribosome
  • RNA: HCV IRESHepatitis C virus internal ribosome entry site

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Supramolecule #1000: IRES-80S ribosome complex

SupramoleculeName: IRES-80S ribosome complex / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: ribosome

SupramoleculeName: ribosome / type: organelle_or_cellular_component / ID: 1 / Details: H. sapiens / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa

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Macromolecule #1: HCV IRES

MacromoleculeName: HCV IRES / type: rna / ID: 1 / Classification: OTHER / Structure: DOUBLE HELIX / 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

BufferpH: 8.1
Details: 20 mM Tris.HCl pH 8.1, 145 mM KCl, 1 mM CaCl2, 5 mM MgCl2, 0.2 mM DTT, 5 mM tobramycin
StainingType: NEGATIVE / Details: native cryo
GridDetails: copper grid
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Software - Name: imagic 5 / Number images used: 24100

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