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- EMDB-2495: Substrate Recruitment Pathways in the Yeast Exosome by Electron M... -

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Basic information

Entry
Database: EMDB / ID: EMD-2495
TitleSubstrate Recruitment Pathways in the Yeast Exosome by Electron Microscopy
Map dataExosome incubated with ssRNA( 16 nucleotides in length)
Sample
  • Sample: Rrp44-Exosome incubated with RNA16
  • 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 / 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
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: 0.0385
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0385
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2495-RNA16-R...

Downloads & links

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Map

FileDownload / File: emd_2495.map.gz / Format: CCP4 / Size: 2.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExosome incubated with ssRNA( 16 nucleotides in length)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.4 Å/pix.
x 90 pix.
= 396. Å		4.4 Å/pix.
x 90 pix.
= 396. Å		4.4 Å/pix.
x 90 pix.
= 396. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.0385 / Movie #1: 0.0385
Minimum - Maximum-0.07484865 - 0.2077371
Average (Standard dev.)-0.00006091 (±0.00848787)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 396.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.44.44.4
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z396.000396.000396.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-0.0750.208-0.000

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Supplemental data

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Sample components

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Entire : Rrp44-Exosome incubated with RNA16

EntireName: Rrp44-Exosome incubated with RNA16
Components
  • Sample: Rrp44-Exosome incubated with RNA16
  • 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: RNA16

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Supramolecule #1000: Rrp44-Exosome incubated with RNA16

SupramoleculeName: Rrp44-Exosome incubated with RNA16 / type: sample / ID: 1000 / Number unique components: 11
Molecular weightExperimental: 350 KDa / Method: Mass spectrum

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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: RNA16

MacromoleculeName: RNA16 / type: rna / ID: 11 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 4 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 at 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 at 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 F20
DateOct 10, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 200 / Average electron dose: 30 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 26788
Final angle assignmentDetails: SPIDER: theta 90 degrees, phi 359.9 degrees

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Atomic model buiding 1

Initial modelPDB ID:

4ifd


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: J / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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