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- EMDB-2827: Structure of the mitoribosome with a hyper-rotated 37S subunit fr... -

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Basic information

Entry
Database: EMDB / ID: EMD-2827
TitleStructure of the mitoribosome with a hyper-rotated 37S subunit from yeast
Map dataSubtomogram average of the mitoribosome with a hyper-rotated 37S subunit from yeast
Sample
  • Sample: Mitoribosome with a hyper-rotated 37S subunit in isolated mitochondria from yeast
  • Complex: 73S mitoribosome
Keywordsribosome / mitoribosome / mitochondria / Mba1 / tomography subtomogram analysis
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 40.0 Å
AuthorsPfeffer S / Woellhaf MW / Herrmann JM / Foerster F
CitationJournal: Nat Commun / Year: 2015
Title: Organization of the mitochondrial translation machinery studied in situ by cryoelectron tomography.
Authors: Stefan Pfeffer / Michael W Woellhaf / Johannes M Herrmann / Friedrich Förster /
Abstract: Whereas the structure and function of cytosolic ribosomes have been studied in great detail, we know surprisingly little about the structural basis of mitochondrial protein synthesis. Here we used ...Whereas the structure and function of cytosolic ribosomes have been studied in great detail, we know surprisingly little about the structural basis of mitochondrial protein synthesis. Here we used cryoelectron tomography and subtomogram analysis to visualize mitoribosomes in isolated yeast mitochondria, avoiding perturbations during ribosomal purification. Most mitoribosomes reside in immediate proximity to the inner mitochondrial membrane, in line with their specialization in the synthesis of hydrophobic membrane proteins. The subtomogram average of membrane-associated mitoribosomes reveals two distinct membrane contact sites, formed by the 21S rRNA expansion segment 96-ES1 and the inner membrane protein Mba1. On the basis of our data, we further hypothesize that Mba1 is not just a passive mitoribosome receptor on the inner membrane, but that it spatially aligns mitoribosomes with the membrane insertion machinery. This study reveals detailed insights into the supramolecular organization of the mitochondrial translation machinery and its association with the inner membrane in translation-competent mitochondria.
History
DepositionNov 25, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseJan 28, 2015-
UpdateJan 28, 2015-
Current statusJan 28, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.7
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2827.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the mitoribosome with a hyper-rotated 37S subunit from yeast
Voxel sizeX=Y=Z: 5.24 Å
Density
Contour LevelBy AUTHOR: 1.7 / Movie #1: 1.7
Minimum - Maximum-8.24723721 - 11.89826107
Average (Standard dev.)0.0 (±0.99999952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 524.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.245.245.24
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z524.000524.000524.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-8.24711.898-0.000

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Supplemental data

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Sample components

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Entire : Mitoribosome with a hyper-rotated 37S subunit in isolated mitocho...

EntireName: Mitoribosome with a hyper-rotated 37S subunit in isolated mitochondria from yeast
Components
  • Sample: Mitoribosome with a hyper-rotated 37S subunit in isolated mitochondria from yeast
  • Complex: 73S mitoribosome

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Supramolecule #1000: Mitoribosome with a hyper-rotated 37S subunit in isolated mitocho...

SupramoleculeName: Mitoribosome with a hyper-rotated 37S subunit in isolated mitochondria from yeast
type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: 73S mitoribosome

SupramoleculeName: 73S mitoribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: D273-10B / synonym: Baker's yeast / Organelle: Mitochondrion

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6 / Details: 20 mM Hepes pH 7.6, 50 mM KCl, 2 mM MgCl2
StainingType: NEGATIVE / Details: No staining
GridDetails: Lacey carbon molybdenum grids
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 60 % / Instrument: FEI VITROBOT MARK IV / Method: Blot time: 4s; blot force: 0

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 7.0 µm / Nominal defocus min: 5.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
DateNov 10, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 100 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 40.0 Å / Resolution method: OTHER / Software - Name: PyTom, tom_toolbox, av3_toolbox / Number subtomograms used: 120
DetailsTomogram reconstruction and template matching against a single particle cryo-EM reconstruction of the 73S yeast mitoribosome were accomplished using PyTom. Tomogram areas corresponding to cross correlation peaks within mitochondria were visually inspected to identify true positive matches. For the retained coordinates, unbinned subtomograms were reconstructed individually from the weighted projections and iteratively aligned using PyTom. Aligned subtomograms were classified using constrained principal component analysis.

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