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- EMDB-2604: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES in a ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2604
TitleKluyveromyces lactis 80S ribosome in complex with CrPV-IRES in a rotated stated with masked 40S
Map dataMap generated through masking the 40S subunit from the EMD-12385 map and performing a re-classification followed by re-refinement both process in the presence of the mask.
Sample
  • Sample: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined
  • Complex: Kluyveromyces lactis 40S ribosome
  • Protein or peptide: CrPV-IRES
Keywordstranslation / ribosomes / eukaryote / initiation
Biological speciesKluyveromyces lactis (yeast) / Cricket paralysis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsFernandez IS / Bai X / Scheres SHW / Ramakrishnan V
CitationJournal: Cell / Year: 2014
Title: Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome.
Authors: Israel S Fernández / Xiao-Chen Bai / Garib Murshudov / Sjors H W Scheres / V Ramakrishnan /
Abstract: The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By ...The cricket paralysis virus internal ribosome entry site (CrPV-IRES) is a folded structure in a viral mRNA that allows initiation of translation in the absence of any host initiation factors. By using recent advances in single-particle electron cryomicroscopy, we have solved the structure of CrPV-IRES bound to the ribosome of the yeast Kluyveromyces lactis in both the canonical and rotated states at overall resolutions of 3.7 and 3.8 Å, respectively. In both states, the pseudoknot PKI of the CrPV-IRES mimics a tRNA/mRNA interaction in the decoding center of the A site of the 40S ribosomal subunit. The structure and accompanying factor-binding data show that CrPV-IRES binding mimics a pretranslocation rather than initiation state of the ribosome. Translocation of the IRES by elongation factor 2 (eEF2) is required to bring the first codon of the mRNA into the A site and to allow the start of translation.
History
DepositionMar 5, 2014-
Header (metadata) releaseApr 9, 2014-
Map releaseMay 21, 2014-
UpdateJun 11, 2014-
Current statusJun 11, 2014Processing 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-4v92
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4v92
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2604.png

Downloads & links

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Map

FileDownload / File: emd_2604.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap generated through masking the 40S subunit from the EMD-12385 map and performing a re-classification followed by re-refinement both process in the presence of the mask.
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.61792678 - 0.99048573
Average (Standard dev.)0.00144863 (±0.02323123)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.6180.9900.001

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Supplemental data

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Sample components

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Entire : Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with ...

EntireName: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined
Components
  • Sample: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined
  • Complex: Kluyveromyces lactis 40S ribosome
  • Protein or peptide: CrPV-IRES

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Supramolecule #1000: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with ...

SupramoleculeName: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES with 40S masked and re-refined
type: sample / ID: 1000 / Number unique components: 2

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Supramolecule #1: Kluyveromyces lactis 40S ribosome

SupramoleculeName: Kluyveromyces lactis 40S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S, SSU RNA 18S
Source (natural)Organism: Kluyveromyces lactis (yeast) / synonym: Kluyveromyces lactis

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Macromolecule #1: CrPV-IRES

MacromoleculeName: CrPV-IRES / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Cricket paralysis virus

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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

Concentration0.1 mg/mL
BufferpH: 6.5
Details: MES-KOH, 40mM K-acetate, 10mM NH4-acetate, 8mM Mg-acetate, 2mM DTT
GridDetails: 400mesh Cu, Quantifoil R2/2
VitrificationCryogen name: PROPANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.0018 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJul 7, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1900 / Average electron dose: 40 e/Å2
Experimental equipment
Model: Titan Krios / 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: 3.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 25969
DetailsMasked 40S area and focused classification and refined in the presence of the mask

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v92:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 2

Initial modelPDB ID:

3u5c
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v92:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 3

Initial modelPDB ID:

3u5d
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v92:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 4

Initial modelPDB ID:

3u5e
PDB Unreleased entry

SoftwareName: Chimera, refmac
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v92:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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