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- EMDB-2599: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES -

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Basic information

Entry
Database: EMDB / ID: EMD-2599
TitleKluyveromyces lactis 80S ribosome in complex with CrPV-IRES
Map dataKluyveromyces lactis 80S ribosome in complex with CrPV-IRES in canonical state
Sample
  • Sample: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES
  • Complex: Kluyveromyces lactis 80S ribosome
  • Protein or peptide: IRES
Keywordseukaryotic / translation / initiation / ribosome / IRES
Biological speciesKluyveromyces lactis (yeast) / Cricket paralysis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFernandez IS / Bai XC / 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 3, 2014-
Header (metadata) releaseApr 9, 2014-
Map releaseMay 14, 2014-
UpdateJul 15, 2015-
Current statusJul 15, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v91
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4v91
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2599.png

Downloads & links

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Map

FileDownload / File: emd_2599.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKluyveromyces lactis 80S ribosome in complex with CrPV-IRES in canonical state
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.84147578 - 1.27515006
Average (Standard dev.)0.00347881 (±0.04991902)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.8411.2750.003

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Supplemental data

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Supplemental map: 40S IntRat WM.map

File40S_IntRat_WM.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Supplemental map: 80S IRES IntRat FINAL.map

File80S_IRES_IntRat_FINAL.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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

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

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

SupramoleculeName: Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES
type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: 1 / Number unique components: 2
Molecular weightExperimental: 3.2 MDa / Theoretical: 3.2 MDa / Method: Sedimentation

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Supramolecule #1: Kluyveromyces lactis 80S ribosome

SupramoleculeName: Kluyveromyces lactis 80S ribosome / type: complex / ID: 1 / Name.synonym: Kluyveromyces lactis 80S ribosome / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Kluyveromyces lactis (yeast)
Molecular weightExperimental: 3.2 MDa / Theoretical: 3.2 MDa

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Macromolecule #1: IRES

MacromoleculeName: 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: 10mM MES-KOH, 10mM NH4 acetate, 40mM K-acetate, 8 mM Mg-acetated, 2mM DTT
GridDetails: 400 mesh R 2/2 Quantifoil grids
VitrificationCryogen name: PROPANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I
Timed resolved state: Vitrified 30 msec after spraying with effector
Method: 2.5

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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: 3.7 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 18132
DetailsRELION movement correction processing used

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Atomic model buiding 1

Initial modelPDB ID:

3b31


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera, refmac
DetailsCoordinates refined with Refmac and checked with COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v91:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 2

Initial modelPDB ID:

2noq


Chain - Chain ID: A
SoftwareName: Chimera, refmac
DetailsCoordinates refined with Refmac and checked with COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v91:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 3

Initial modelPDB ID:

3u5c
PDB Unreleased entry


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U / Chain - #21 - Chain ID: V / Chain - #22 - Chain ID: W / Chain - #23 - Chain ID: X / Chain - #24 - Chain ID: Y / Chain - #25 - Chain ID: Z / Chain - #26 - Chain ID: a / Chain - #27 - Chain ID: b / Chain - #28 - Chain ID: c / Chain - #29 - Chain ID: d / Chain - #30 - Chain ID: e / Chain - #31 - Chain ID: f / Chain - #32 - Chain ID: g
SoftwareName: Chimera, refmac
DetailsCoordinates refined with Refmac and checked with COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v91:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 4

Initial modelPDB ID:

3u5e
PDB Unreleased entry


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L / Chain - #12 - Chain ID: M / Chain - #13 - Chain ID: N / Chain - #14 - Chain ID: O / Chain - #15 - Chain ID: P / Chain - #16 - Chain ID: Q / Chain - #17 - Chain ID: R / Chain - #18 - Chain ID: S / Chain - #19 - Chain ID: T / Chain - #20 - Chain ID: U / Chain - #21 - Chain ID: V / Chain - #22 - Chain ID: W / Chain - #23 - Chain ID: X / Chain - #24 - Chain ID: Y / Chain - #25 - Chain ID: Z / Chain - #26 - Chain ID: a / Chain - #27 - Chain ID: b / Chain - #28 - Chain ID: c / Chain - #29 - Chain ID: d / Chain - #30 - Chain ID: e / Chain - #31 - Chain ID: f / Chain - #32 - Chain ID: g / Chain - #33 - Chain ID: h / Chain - #34 - Chain ID: i / Chain - #35 - Chain ID: j / Chain - #36 - Chain ID: k / Chain - #37 - Chain ID: l / Chain - #38 - Chain ID: m / Chain - #39 - Chain ID: n / Chain - #40 - Chain ID: o / Chain - #41 - Chain ID: p
SoftwareName: Chimera, refmac
DetailsCoordinates refined with Refmac and checked with COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v91:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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Atomic model buiding 5

Initial modelPDB ID:

3u5d
PDB Unreleased entry


Chain - #0 - Chain ID: 1 / Chain - #1 - Chain ID: 3 / Chain - #2 - Chain ID: 4
SoftwareName: Chimera, refmac
DetailsCoordinates refined with Refmac and checked with COOT
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 60 / Target criteria: R-factor, FSC
Output model

PDB-4v91:
Kluyveromyces lactis 80S ribosome in complex with CrPV-IRES

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