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- EMDB-5329: Cryo EM reconstruction of mammalian 80S ribosome in rotated PRE s... -

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Basic information

Entry
Database: EMDB / ID: EMD-5329
TitleCryo EM reconstruction of mammalian 80S ribosome in rotated PRE state 2
Map data80S mammalian ribosome
Sample
  • Sample: 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver
  • Complex: 80S ribosome
Keywords80S ribosome / mammalian / elongation cycle / tRNA
Function / homology
Function and homology information


SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit ...SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / structural constituent of ribosome / translation / RNA binding / nucleus / cytosol
Similarity search - Function
Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / : / Ribosomal protein L5, conserved site / Ribosomal protein L5 signature. ...Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / : / Ribosomal protein L5, conserved site / Ribosomal protein L5 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L5 / Ribosomal protein L5, C-terminal / ribosomal L5P family C-terminus / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily
Similarity search - Domain/homology
Large ribosomal subunit protein uL1A / Large ribosomal subunit protein uL5B
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.6 Å
AuthorsBudkevich T / Giesebrecht J / Altman R / Munro J / Mielke T / Nierhaus K / Blanchard S / Spahn C
CitationJournal: Mol Cell / Year: 2011
Title: Structure and dynamics of the mammalian ribosomal pretranslocation complex.
Authors: Tatyana Budkevich / Jan Giesebrecht / Roger B Altman / James B Munro / Thorsten Mielke / Knud H Nierhaus / Scott C Blanchard / Christian M T Spahn /
Abstract: Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to ...Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.
History
DepositionJul 12, 2011-
Header (metadata) releaseJul 28, 2011-
Map releaseNov 9, 2011-
UpdateMar 6, 2013-
Current statusMar 6, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1000
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1000
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j0q
  • Surface level: 1000
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j0q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5329_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5329.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation80S mammalian ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.52 Å/pix.
x 180 pix.
= 453.6 Å		2.52 Å/pix.
x 180 pix.
= 453.6 Å		2.52 Å/pix.
x 180 pix.
= 453.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.52 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1000.0 / Movie #1: 1000
Minimum - Maximum-2542.372802729999876 - 17103.236328129998583
Average (Standard dev.)275.671539310000014 (±1215.576416019999897)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-89
Dimensions180180180
Spacing180180180
CellA=B=C: 453.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.522.522.52
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z453.600453.600453.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-90-90-89
NC/NR/NS180180180
D min/max/mean-2542.37317103.236275.672

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Supplemental data

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Sample components

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Entire : 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver

EntireName: 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver
Components
  • Sample: 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver
  • Complex: 80S ribosome

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Supramolecule #1000: 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver

SupramoleculeName: 80S-PRE-rotated-2 complex from rabbit (Oryctolagus cuniculus) liver
type: sample / ID: 1000 / Details: Sample was re-associated from subunits / Oligomeric state: monomer / Number unique components: 5
Molecular weightExperimental: 4 MDa / Method: Sedimentation

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: LSU 60S, SSU 40S
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: liver
Molecular weightTheoretical: 4 MDa

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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: 7.5
GridDetails: Quantifoil grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: OTHER / Details: Vitrification instrument: VitroBot (FEI)
Method: Flash-frozen in liquid ethane on carbon coated Quantifoil grids

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 77 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
DateOct 17, 2006
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.7 µm / Number real images: 87 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 65520 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder: Cartridge / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: By defocus groups
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Several rounds of multireference 3D projection refinement
Number images used: 23347

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