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- EMDB-5527: Reconstruction of the deltaGCC mutant HCV IRES bound to rabbit 40... -

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Basic information

Entry
Database: EMDB / ID: EMD-5527
TitleReconstruction of the deltaGCC mutant HCV IRES bound to rabbit 40S ribosomal subunit
Map dataReconstruction of mutant HCV IRES bound to 40S
Sample
  • Sample: mutant HCV IRES bound to 40S
  • Complex: Rabbit 40S ribosomal subunit
  • RNA: hepatitis C virus internal ribosome entry site
KeywordsHCV IRES / 40S
Biological speciesOryctolagus cuniculus (rabbit) / Hepatitis C virus
Methodsingle particle reconstruction / cryo EM / Resolution: 17.5 Å
AuthorsVollmar BS / Shi D / Filbin ME / Kieft JS / Gonen T
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.
Authors: Megan E Filbin / Breanna S Vollmar / Dan Shi / Tamir Gonen / Jeffrey S Kieft /
Abstract: The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have ...The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders.
History
DepositionNov 2, 2012-
Header (metadata) releaseNov 21, 2012-
Map releaseApr 17, 2013-
UpdateApr 17, 2013-
Current statusApr 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.25
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5527_1.tif

Downloads & links

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Map

FileDownload / File: emd_5527.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mutant HCV IRES bound to 40S
Voxel sizeX=Y=Z: 2.66 Å
Density
Contour LevelBy AUTHOR: 2.25 / Movie #1: 2.25
Minimum - Maximum-6.43432808 - 9.639652249999999
Average (Standard dev.)0.0 (±1.00000012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 399.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.662.662.66
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z399.000399.000399.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-6.4349.6400.000

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Supplemental data

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Sample components

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Entire : mutant HCV IRES bound to 40S

EntireName: mutant HCV IRES bound to 40S
Components
  • Sample: mutant HCV IRES bound to 40S
  • Complex: Rabbit 40S ribosomal subunit
  • RNA: hepatitis C virus internal ribosome entry site

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Supramolecule #1000: mutant HCV IRES bound to 40S

SupramoleculeName: mutant HCV IRES bound to 40S / type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Rabbit 40S ribosomal subunit

SupramoleculeName: Rabbit 40S ribosomal subunit / type: complex / ID: 1 / Name.synonym: 40S ribosome / Details: Mutant HCV IRES bound to 40S / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte

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Macromolecule #1: hepatitis C virus internal ribosome entry site

MacromoleculeName: hepatitis C virus internal ribosome entry site / type: rna / ID: 1 / Name.synonym: HCV IRES
Details: Deletion of GCC (82-84) of HCV IRES (nucleotides 40-372)
Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Hepatitis C virus / synonym: HCV

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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 Tris-HCl, pH 7.4, 200 mM KOAc, 2.5 mM MgCl2, 2 mM DTT, 40 mM KCl
GridDetails: Quantifoil R1.2/1.3 400 mesh copper grids with holey carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot and plunge into liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 117293 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateMar 19, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 1790 / Average electron dose: 15 e/Å2
Details: Used 2x binned images yielding a pixel size of 2.66 A/pixel.
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.5 Å / Resolution method: OTHER / Software - Name: FREALIGN / Number images used: 29920
DetailsParticles selected manually using Electron Micrographe Utility (cryoem.ucsf.edu)

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