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- EMDB-2179: Electron cryo-microscopy of C. thermophilum RAC bound to the 80S ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2179
TitleElectron cryo-microscopy of C. thermophilum RAC bound to the 80S ribosome
Map dataReconstruction of the reconstituted complex of C. thermophilum RAC and C. thermophilum 80S ribosomes (Low-pass filtered between 18-20 A)
Sample
  • Sample: Ribosome-associated complex (RAC) bound to 80S ribosome
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: Ribosome-associated complex
Keywordsribosome-associated complex / RAC / ribosome / 80S / chaperone / co-translational folding
Biological speciesChaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.6 Å
AuthorsLeidig C / Bange G / Kopp J / Amlacher S / Aravind A / Wickles S / Witte G / Hurt E / Beckmann R / Sinning I
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Structural characterization of a eukaryotic chaperone--the ribosome-associated complex.
Authors: Christoph Leidig / Gert Bange / Jürgen Kopp / Stefan Amlacher / Ajay Aravind / Stephan Wickles / Gregor Witte / Ed Hurt / Roland Beckmann / Irmgard Sinning /
Abstract: Ribosome-associated chaperones act in early folding events during protein synthesis. Structural information is available for prokaryotic chaperones (such as trigger factor), but structural ...Ribosome-associated chaperones act in early folding events during protein synthesis. Structural information is available for prokaryotic chaperones (such as trigger factor), but structural understanding of these processes in eukaryotes lags far behind. Here we present structural analyses of the eukaryotic ribosome-associated complex (RAC) from Saccharomyces cerevisiae and Chaetomium thermophilum, consisting of heat-shock protein 70 (Hsp70) Ssz1 and the Hsp40 Zuo1. RAC is an elongated complex that crouches over the ribosomal tunnel exit and seems to be stabilized in a distinct conformation by expansion segment ES27. A unique α-helical domain in Zuo1 mediates ribosome interaction of RAC near the ribosomal proteins L22e and L31e and ribosomal RNA helix H59. The crystal structure of the Ssz1 ATPase domain bound to ATP-Mg²⁺ explains its catalytic inactivity and suggests that Ssz1 may act before the RAC-associated chaperone Ssb. Our study offers insights into the interplay between RAC, the ER membrane-integrated Hsp40-type protein ERj1 and the signal-recognition particle.
History
DepositionAug 20, 2012-
Header (metadata) releaseAug 29, 2012-
Map releaseFeb 27, 2013-
UpdateFeb 27, 2013-
Current statusFeb 27, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 160
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 160
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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Map

FileDownload / File: emd_2179.map.gz / Format: CCP4 / Size: 39.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the reconstituted complex of C. thermophilum RAC and C. thermophilum 80S ribosomes (Low-pass filtered between 18-20 A)
Voxel sizeX=Y=Z: 2.1 Å
Density
Contour LevelBy AUTHOR: 160.0 / Movie #1: 160
Minimum - Maximum-1450.795410160000074 - 3006.467529300000024
Average (Standard dev.)16.64499855 (±275.968292239999982)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-109-109-109
Dimensions219219219
Spacing219219219
CellA=B=C: 459.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z219219219
origin x/y/z0.0000.0000.000
length x/y/z459.900459.900459.900
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS-109-109-109
NC/NR/NS219219219
D min/max/mean-1450.7953006.46816.645

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Supplemental data

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Sample components

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Entire : Ribosome-associated complex (RAC) bound to 80S ribosome

EntireName: Ribosome-associated complex (RAC) bound to 80S ribosome
Components
  • Sample: Ribosome-associated complex (RAC) bound to 80S ribosome
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: Ribosome-associated complex

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Supramolecule #1000: Ribosome-associated complex (RAC) bound to 80S ribosome

SupramoleculeName: Ribosome-associated complex (RAC) bound to 80S ribosome
type: sample / ID: 1000
Details: Sample was reconstituted from the purified components
Oligomeric state: One RAC binds to one ribosome / Number unique components: 2
Molecular weightTheoretical: 3.2 MDa

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Chaetomium thermophilum (fungus) / Location in cell: cytosol
Molecular weightTheoretical: 3.1 MDa

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Macromolecule #1: Ribosome-associated complex

MacromoleculeName: Ribosome-associated complex / type: protein_or_peptide / ID: 1 / Name.synonym: RAC / Recombinant expression: No
Source (natural)Organism: Chaetomium thermophilum (fungus) / Location in cell: cytosol
Molecular weightTheoretical: 110 KDa

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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
Details: 20 mM Tris-HCl, 100 mM KOAc, 100 mM sucrose, 7.5 mM Mg(OAc)2, 2 mM DTT
GridDetails: carbon supported
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III / Method: Blot for 3.5 s before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsFinal magnification of the object on the CCD image is 148721
DateJul 6, 2011
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 6954 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Particle groups with similar defocus
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Sorting for ribosome conformation and ligand presence was performed
Number images used: 26038
DetailsParticles selected by signature. Refinement with spider.

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