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- EMDB-1197: A mechanical explanation of RNA pseudoknot function in programmed... -

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Entry
Database: EMDB / ID: EMD-1197
TitleA mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting.
Map data3D reconstruction of IBV pseudoknot-stalled rabbit reticulocyte ribosome containing tRNA, eEF2, pseudoknot and both ribosomal subunits.
Sample
  • Sample: Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudoknot with tRNA and eEF2
  • Complex: Small subunit
  • Complex: Large subunit
  • RNA: Message with pseudoknot
  • RNA: P-site transfer RNA
  • Protein or peptide: Elongation translocase
Biological speciesInfectious bronchitis virus / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.2 Å
AuthorsNamy O / Moran SJ / Stuart DI / Gilbert RJC / Brierley I
CitationJournal: Nature / Year: 2006
Title: A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting.
Authors: Olivier Namy / Stephen J Moran / David I Stuart / Robert J C Gilbert / Ian Brierley /
Abstract: The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal ...The triplet-based genetic code requires that translating ribosomes maintain the reading frame of a messenger RNA faithfully to ensure correct protein synthesis. However, in programmed -1 ribosomal frameshifting, a specific subversion of frame maintenance takes place, wherein the ribosome is forced to shift one nucleotide backwards into an overlapping reading frame and to translate an entirely new sequence of amino acids. This process is indispensable in the replication of numerous viral pathogens, including HIV and the coronavirus associated with severe acute respiratory syndrome, and is also exploited in the expression of several cellular genes. Frameshifting is promoted by an mRNA signal composed of two essential elements: a heptanucleotide 'slippery' sequence and an adjacent mRNA secondary structure, most often an mRNA pseudoknot. How these components operate together to manipulate the ribosome is unknown. Here we describe the observation of a ribosome-mRNA pseudoknot complex that is stalled in the process of -1 frameshifting. Cryoelectron microscopic imaging of purified mammalian 80S ribosomes from rabbit reticulocytes paused at a coronavirus pseudoknot reveals an intermediate of the frameshifting process. From this it can be seen how the pseudoknot interacts with the ribosome to block the mRNA entrance channel, compromising the translocation process and leading to a spring-like deformation of the P-site transfer RNA. In addition, we identify movements of the likely eukaryotic ribosomal helicase and confirm a direct interaction between the translocase eEF2 and the P-site tRNA. Together, the structural changes provide a mechanical explanation of how the pseudoknot manipulates the ribosome into a different reading frame.
History
DepositionFeb 28, 2006-
Header (metadata) releaseFeb 28, 2006-
Map releaseMay 11, 2006-
UpdateSep 2, 2011-
Current statusSep 2, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 19.250294705
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 19.250294705
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1197.gif

Downloads & links

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Map

FileDownload / File: emd_1197.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of IBV pseudoknot-stalled rabbit reticulocyte ribosome containing tRNA, eEF2, pseudoknot and both ribosomal subunits.
Voxel sizeX=Y=Z: 3.33 Å
Density
Contour LevelBy EMDB: 10.0 / Movie #1: 19.2502947
Minimum - Maximum-161.699999999999989 - 200.0
Average (Standard dev.)-5.50367 (±9.305809999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-80-80-79
Dimensions160160160
Spacing160160160
CellA=B=C: 532.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.333.333.33
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z532.800532.800532.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-80-80-79
NX/NY/NZ160160160
MAP C/R/S213
start NC/NR/NS-80-80-79
NC/NR/NS160160160
D min/max/mean-161.700200.000-5.504

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Supplemental data

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Sample components

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Entire : Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudo...

EntireName: Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudoknot with tRNA and eEF2
Components
  • Sample: Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudoknot with tRNA and eEF2
  • Complex: Small subunit
  • Complex: Large subunit
  • RNA: Message with pseudoknot
  • RNA: P-site transfer RNA
  • Protein or peptide: Elongation translocase

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Supramolecule #1000: Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudo...

SupramoleculeName: Rabbit reticuloyte ribosome stalled on IBV mRNA containing pseudoknot with tRNA and eEF2
type: sample / ID: 1000
Oligomeric state: One each of 40S, 60S, mRNA, eEF2 and P-site tRNA
Number unique components: 5
Molecular weightExperimental: 3.5 MDa / Theoretical: 3.5 MDa

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Supramolecule #1: Small subunit

SupramoleculeName: Small subunit / type: complex / ID: 1 / Name.synonym: 40S / Ribosome-details: ribosome-eukaryote: SSU 40S

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Supramolecule #2: Large subunit

SupramoleculeName: Large subunit / type: complex / ID: 2 / Name.synonym: 60S / Ribosome-details: ribosome-eukaryote: LSU 60S

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Macromolecule #1: Message with pseudoknot

MacromoleculeName: Message with pseudoknot / type: rna / ID: 1 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Infectious bronchitis virus / synonym: IBV

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Macromolecule #3: P-site transfer RNA

MacromoleculeName: P-site transfer RNA / type: rna / ID: 3 / Name.synonym: tRNA / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit

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Macromolecule #2: Elongation translocase

MacromoleculeName: Elongation translocase / type: protein_or_peptide / ID: 2 / Name.synonym: eEF2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: Spleen / Cell: Reticulocytes / Location in cell: Cytosol

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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

GridDetails: 300 mesh gold w/ lacey carbon
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 100 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 3.33 µm / Number real images: 31 / Average electron dose: 2 e/Å2 / Od range: 5 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe particles were selected in a semi-automated fashion using BOXER (EMAN suite)
CTF correctionDetails: By micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER and GAP
Details: Deposited map is composite of computationally-separated subunits and bound co-factors from the final map.
Number images used: 17672
Final angle assignmentDetails: SPIDER

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Atomic model buiding 1

SoftwareName: URO
DetailsProtocol: Rigid body. Domains manually docked in O and refined in URO
RefinementProtocol: RIGID BODY FIT / Target criteria: CC

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