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- EMDB-2501: Substrate Recruitment Pathways in the Yeast Exosome by Electron M... -

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Basic information

Entry
Database: EMDB / ID: EMD-2501
TitleSubstrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy
Map dataExosome with tRNA
Sample
  • Sample: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE
  • Protein or peptide: x 10 types
  • RNA: x 1 types
Keywordsexosome / RNA degradation route
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 24.0 Å
AuthorsLiu J-J / Bratkowski MA / Liu XQ / Niu C-Y / Ke AL / Wang H-W
CitationJournal: Nat Struct Mol Biol / Year: 2014
Title: Visualization of distinct substrate-recruitment pathways in the yeast exosome by EM.
Authors: Jun-Jie Liu / Matthew A Bratkowski / Xueqi Liu / Chu-Ya Niu / Ailong Ke / Hong-Wei Wang /
Abstract: The eukaryotic exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3'-to-5' exo- and endo-RNase activities. RNA substrates have ...The eukaryotic exosome is a multisubunit complex typically composed of a catalytically inactive core and the Rrp44 protein, which contains 3'-to-5' exo- and endo-RNase activities. RNA substrates have been shown to be recruited through the core to reach Rrp44's exo-RNase (EXO) site. Using single-particle EM and biochemical analysis, we provide visual evidence that two distinct substrate-recruitment pathways exist. In the through-core route, channeling of the single-stranded substrates from the core to Rrp44 induces a characteristic conformational change in Rrp44. In the alternative direct-access route, this conformational change does not take place, and the RNA substrate is visualized to avoid the core and enter Rrp44's EXO site directly. Our results provide mechanistic explanations for several RNA processing scenarios by the eukaryotic exosome and indicate substrate-specific modes of degradation by this complex.
History
DepositionOct 23, 2013-
Header (metadata) releaseOct 30, 2013-
Map releaseDec 25, 2013-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.032
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.032
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2501.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExosome with tRNA
Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.032
Minimum - Maximum-0.06066232 - 0.13091946
Average (Standard dev.)0.00065984 (±0.00929574)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 360.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0610.1310.001

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Supplemental data

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Sample components

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Entire : Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE

EntireName: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE
Components
  • Sample: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE
  • Protein or peptide: Rrp44
  • Protein or peptide: Rrp43
  • Protein or peptide: Rrp4
  • Protein or peptide: Csl4
  • Protein or peptide: Rrp45
  • Protein or peptide: Rrp46-TAP
  • Protein or peptide: Rrp41
  • Protein or peptide: Rrp42
  • Protein or peptide: Mtr3
  • Protein or peptide: Rrp40
  • RNA: methionine tRNA

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Supramolecule #1000: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE

SupramoleculeName: Rrp44-Exosome with tRNA by Cryo-EMtRNA-RE / type: sample / ID: 1000 / Number unique components: 11
Molecular weightExperimental: 350 KDa

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Macromolecule #1: Rrp44

MacromoleculeName: Rrp44 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 110 KDa

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Macromolecule #2: Rrp43

MacromoleculeName: Rrp43 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 40 KDa

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Macromolecule #3: Rrp4

MacromoleculeName: Rrp4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 40 KDa

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Macromolecule #4: Csl4

MacromoleculeName: Csl4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #5: Rrp45

MacromoleculeName: Rrp45 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #6: Rrp46-TAP

MacromoleculeName: Rrp46-TAP / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #7: Rrp41

MacromoleculeName: Rrp41 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #8: Rrp42

MacromoleculeName: Rrp42 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #9: Mtr3

MacromoleculeName: Mtr3 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #10: Rrp40

MacromoleculeName: Rrp40 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightExperimental: 30 KDa

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Macromolecule #11: methionine tRNA

MacromoleculeName: methionine tRNA / type: rna / ID: 11 / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 25 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

Concentration2 mg/mL
BufferpH: 8 / Details: 150mM NaCl, 50mM Tris-HCL,1mM DTT, 2mM MgCl2
GridDetails: Quantifoil grids (2/2) with2~3 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK IV
Method: 4 ul of the reaction solution was then applied to glow-discharged C-flat grids (1.2/1.3) covered with a layer of continuous carbon with a thickness of ~4nm. The grids were then blotted and ...Method: 4 ul of the reaction solution was then applied to glow-discharged C-flat grids (1.2/1.3) covered with a layer of continuous carbon with a thickness of ~4nm. The grids were then blotted and plunged into liquid ethane in a FEI Vitrobot Mark IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 85 K / Average: 82 K
DateJun 10, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 1200 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: both each particle and micrograph
Final angle assignmentDetails: RELION: theta 45 degrees, phi 180 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: eman, imagic, relion / Number images used: 28800
DetailsMicrographs of RE were collected using the AutoEMation software (Lei and Frank, 2005) installed on the microscope at low-dose condition with a dose of ~20 electron/A2 and a defocus value ranging from -1.2 to -4 um. The micrographs were collected on a FEI Eagle CCD camera with a pixel size of 1.5 A.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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