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- EMDB-1708: Cryo-EM structure of S. cerevisiae 10-subunit exosome -

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Basic information

Entry
Database: EMDB / ID: EMD-1708
TitleCryo-EM structure of S. cerevisiae 10-subunit exosome
Map dataCryo-EM map of S. cerevisiae 10-subunit exosome
Sample
  • Sample: S. cerevisiae 10-subunit exosome
  • Protein or peptide: Rrp41
  • Protein or peptide: Rrp42
  • Protein or peptide: Rrp43
  • Protein or peptide: Rrp44
  • Protein or peptide: Rrp45
  • Protein or peptide: Rrp46
  • Protein or peptide: Mtr3
  • Protein or peptide: Rrp4
  • Protein or peptide: Rrp40
  • Protein or peptide: Csl4
Keywordsexosome / cryo-EM / RNA quality control / exoribonuclease
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsMalet H / Topf M / Clare DK / Ebert J / Bonneau F / Basquin J / Drazkowska K / Tomecki R / Dziembowski A / Conti E ...Malet H / Topf M / Clare DK / Ebert J / Bonneau F / Basquin J / Drazkowska K / Tomecki R / Dziembowski A / Conti E / Saibil HR / Lorentzen E
CitationJournal: EMBO Rep / Year: 2010
Title: RNA channelling by the eukaryotic exosome.
Authors: Hélène Malet / Maya Topf / Daniel K Clare / Judith Ebert / Fabien Bonneau / Jerome Basquin / Karolina Drazkowska / Rafal Tomecki / Andrzej Dziembowski / Elena Conti / Helen R Saibil / Esben Lorentzen /
Abstract: The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models ...The eukaryotic exosome is a key nuclease for the degradation, processing and quality control of a wide variety of RNAs. Here, we report electron microscopic reconstructions and pseudo-atomic models of the ten-subunit Saccharomyces cerevisiae exosome in the unbound and RNA-bound states. In the RNA-bound structures, extra density that is visible at the entry and exit sites of the exosome channel indicates that a substrate-threading mechanism is used by the eukaryotic exosome. This channelling mechanism seems to be conserved in exosome-like complexes from all domains of life, and might have been present in the most recent common ancestor.
History
DepositionMar 9, 2010-
Header (metadata) releaseApr 9, 2010-
Map releaseDec 22, 2010-
UpdateAug 29, 2012-
Current statusAug 29, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1708apoe...

Downloads & links

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Map

FileDownload / File: emd_1708.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of S. cerevisiae 10-subunit exosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.6 Å/pix.
x 128 pix.
= 332.8 Å		2.6 Å/pix.
x 128 pix.
= 332.8 Å		2.6 Å/pix.
x 128 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.39733 - 10.0
Average (Standard dev.)0.0229963 (±0.293644)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 332.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-61-61-60
NX/NY/NZ122122122
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.39710.0000.023

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Supplemental data

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Sample components

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Entire : S. cerevisiae 10-subunit exosome

EntireName: S. cerevisiae 10-subunit exosome
Components
  • Sample: S. cerevisiae 10-subunit exosome
  • Protein or peptide: Rrp41
  • Protein or peptide: Rrp42
  • Protein or peptide: Rrp43
  • Protein or peptide: Rrp44
  • Protein or peptide: Rrp45
  • Protein or peptide: Rrp46
  • Protein or peptide: Mtr3
  • Protein or peptide: Rrp4
  • Protein or peptide: Rrp40
  • Protein or peptide: Csl4

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Supramolecule #1000: S. cerevisiae 10-subunit exosome

SupramoleculeName: S. cerevisiae 10-subunit exosome / type: sample / ID: 1000 / Oligomeric state: Heterodecamer / Number unique components: 10
Molecular weightExperimental: 400 KDa / Theoretical: 400 KDa / Method: Gel filtration

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Macromolecule #1: Rrp41

MacromoleculeName: Rrp41 / type: protein_or_peptide / ID: 1 / Name.synonym: Rrp41 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 27.6 KDa / Theoretical: 27.6 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #2: Rrp42

MacromoleculeName: Rrp42 / type: protein_or_peptide / ID: 2 / Name.synonym: Rrp42 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 29.1 KDa / Theoretical: 29.1 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #3: Rrp43

MacromoleculeName: Rrp43 / type: protein_or_peptide / ID: 3 / Name.synonym: Rrp43 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 44 KDa / Theoretical: 44 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #4: Rrp44

MacromoleculeName: Rrp44 / type: protein_or_peptide / ID: 4 / Name.synonym: Dis3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 113.7 KDa / Theoretical: 113.7 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #5: Rrp45

MacromoleculeName: Rrp45 / type: protein_or_peptide / ID: 5 / Name.synonym: Rrp45 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 34 KDa / Theoretical: 34 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #6: Rrp46

MacromoleculeName: Rrp46 / type: protein_or_peptide / ID: 6 / Name.synonym: Rrp46 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 24.4 KDa / Theoretical: 24.4 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #7: Mtr3

MacromoleculeName: Mtr3 / type: protein_or_peptide / ID: 7 / Name.synonym: Mtr3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 27.3 KDa / Theoretical: 27.3 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #8: Rrp4

MacromoleculeName: Rrp4 / type: protein_or_peptide / ID: 8 / Name.synonym: Rrp4 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 39.4 KDa / Theoretical: 39.4 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #9: Rrp40

MacromoleculeName: Rrp40 / type: protein_or_peptide / ID: 9 / Name.synonym: Rrp40 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 26.6 KDa / Theoretical: 226.6 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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Macromolecule #10: Csl4

MacromoleculeName: Csl4 / type: protein_or_peptide / ID: 10 / Name.synonym: Csl4
Details: Present in substochiometric amount compared to the other subunits
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YJM789 / synonym: Baker's Yeast / Location in cell: Nucleus, cytoplasm
Molecular weightExperimental: 31.6 KDa / Theoretical: 31.6 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (DE3) (bacteria) / Recombinant plasmid: pET

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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

Concentration0.3 mg/mL
BufferpH: 7.5 / Details: 20 mM Tris-Hcl pH 7.5, 50 mM NaCl, 1 mM DTT
GridDetails: C-flat grid CF-2/2-4C-100
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Manual plunger / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Legacy - Electron beam tilt params: 0
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 250 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 121000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 121000
Sample stageSpecimen holder: Gatan helium / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC-5, SPIDER / Number images used: 15000

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Atomic model buiding 1

Initial modelPDB ID:

2nn6


Chain - #0 - Chain ID: C / Chain - #1 - Chain ID: D / Chain - #2 - Chain ID: E / Chain - #3 - Chain ID: F / Chain - #4 - Chain ID: H
SoftwareName: Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Flexible fitting of rigid bodies. Homology models of yeast subunits have been calculated using MODELLER from the chains C, D, E, F and H of the PDB entry 2NN6. Resulting homology models have been used for flexible fitting into the cryo-EM map.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation, energy

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Atomic model buiding 2

Initial modelPDB ID:

2wp8


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: J
SoftwareName: Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Flexible fitting of rigid bodies
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation, energy

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Atomic model buiding 3

Initial modelPDB ID:

2ja9


Chain - Chain ID: A
SoftwareName: Flex-EM
DetailsPDBEntryID_givenInChain. Protocol: Flexible fitting of rigid body
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation, energy

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