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- EMDB-2522: Substrate Recruitment Pathways in the Yeast Exosome by Electron M... -

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Basic information

Entry
Database: EMDB / ID: EMD-2522
TitleSubstrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy
Map dataExosome incubated with HDV
Sample
  • Sample: HDV-RE
  • Protein or peptide: Rrp44
Keywordsexosome / RNA degradation route
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 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
DepositionNov 26, 2013-
Header (metadata) releaseDec 11, 2013-
Map releaseJan 8, 2014-
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: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

EMD-2522-RE-...

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Map

FileDownload / File: emd_2522.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExosome incubated with HDV
Voxel sizeX=Y=Z: 4.4 Å
Density
Contour LevelBy EMDB: 3.5 / Movie #1: 2
Minimum - Maximum-5.49889565 - 10.98099041
Average (Standard dev.)0.0 (±0.99999809)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z281.600281.600281.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-5.49910.9810.000

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Supplemental data

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Sample components

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Entire : HDV-RE

EntireName: HDV-RE
Components
  • Sample: HDV-RE
  • Protein or peptide: Rrp44

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Supramolecule #1000: HDV-RE

SupramoleculeName: HDV-RE / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 350 KDa / Method: Mass spectrometry

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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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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8 / Details: 150mM NaCl, 50mM Tris-HCL,1mM DTT
StainingType: NEGATIVE
Details: All samples were diluted to a final concentration of ~80 nM of the exosome in the non-digestive buffer and negatively stained in 2% (w/v) uranyl acetate solution following the standard deep ...Details: All samples were diluted to a final concentration of ~80 nM of the exosome in the non-digestive buffer and negatively stained in 2% (w/v) uranyl acetate solution following the standard deep stain procedure on holey-carbon coated EM copper grids covered with a thin layer of continuous carbon
GridDetails: 300 mesh continus carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: OTHER
DateOct 10, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

Final angle assignmentDetails: SPIDER: theta 90 degrees, phi 359.9 degrees
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: OTHER / Software - Name: Spider / Number images used: 20000

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