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- EMDB-3232: The architecture of the S. pombe CCR4-NOT complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3232
TitleThe architecture of the S. pombe CCR4-NOT complex
Map dataCryo-EM reconstruction of the CCR4-NOT complex
Sample
  • Sample: S. pombe CCR4-NOT complex
  • Protein or peptide: CCR4-NOT complex
KeywordsCCR4-NOT / cryo-electron microscopy / RNA decay / deadenylation / single-particle 3D reconstruction
Biological speciesSchizosaccharomyces pombe (fission yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsUkleja M / Cuellar J / Siwaszek A / Kasprzak JM / Czarnocki-Cieciura M / Bujnicki J / Dziembowski A / Valpuesta JM
CitationJournal: Nat Commun / Year: 2016
Title: The architecture of the Schizosaccharomyces pombe CCR4-NOT complex.
Authors: Marta Ukleja / Jorge Cuellar / Aleksandra Siwaszek / Joanna M Kasprzak / Mariusz Czarnocki-Cieciura / Janusz M Bujnicki / Andrzej Dziembowski / Jose M Valpuesta /
Abstract: CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic ...CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic information such as poly(A) tail shortening, transcription regulation, nuclear export and protein degradation, there is only fragmentary information available on some of its nine subunits. Here we show a comprehensive structural characterization of the native CCR4-NOT complex from Schizosaccharomyces pombe. Our cryo-EM 3D reconstruction of the complex, combined with techniques such as immunomicroscopy, RNA-nanogold labelling, docking of the available high-resolution structures and models of different subunits and domains, allow us to propose its full molecular architecture. We locate all functionally defined domains endowed with deadenylating and ubiquitinating activities, the nucleus-specific RNA-interacting subunit Mmi1, as well as surfaces responsible for protein-protein interactions. This information provides insight into cooperation of the different CCR4-NOT complex functions.
History
DepositionNov 5, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseJun 1, 2016-
UpdateJun 22, 2016-
Current statusJun 22, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.348
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.348
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3232_ccr...

Downloads & links

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Map

FileDownload / File: emd_3232.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the CCR4-NOT complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.2 Å/pix.
x 70 pix.
= 294. Å		4.2 Å/pix.
x 70 pix.
= 294. Å		4.2 Å/pix.
x 70 pix.
= 294. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 4.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.348 / Movie #1: 0.348
Minimum - Maximum-0.00219599 - 10.73409462
Average (Standard dev.)0.09819236 (±0.67935294)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 294.0 Å
α=β=γ: 89.999 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z294.000294.000294.000
α/β/γ89.99989.99989.999
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-0.00210.7340.098

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Supplemental data

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Sample components

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Entire : S. pombe CCR4-NOT complex

EntireName: S. pombe CCR4-NOT complex
Components
  • Sample: S. pombe CCR4-NOT complex
  • Protein or peptide: CCR4-NOT complex

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Supramolecule #1000: S. pombe CCR4-NOT complex

SupramoleculeName: S. pombe CCR4-NOT complex / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 700 MDa

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Macromolecule #1: CCR4-NOT complex

MacromoleculeName: CCR4-NOT complex / type: protein_or_peptide / ID: 1
Details: CCR4-NOT complex purified from the endogenous expression of S. pombe cells. The complex is composed of Not1, Not2, Not3, Not4, Caf1, Caf40, Ccr4, Mmi1 subunits
Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast) / synonym: Fission yeast / Location in cell: nucleus/cytoplasm
Molecular weightTheoretical: 700 MDa

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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

Concentration0.2 mg/mL
BufferpH: 7.4 / Details: 10 mM HEPES, 500 mM NaCl, ~3% glycerol
GridDetails: Quantifoil R 1.2/ R1.3 300 mesh grids; ref. Q09684
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: LEICA EM CPC
Method: Aliquots (5 ul) of purified concentrated CCR4-NOT were incubated (2-5 min) with the grid, blotted and plunged into a liquid ethane chamber.

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMay 20, 2014
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 4.2 µm / Number real images: 600 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Cs: 2.0 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailssingle-particle 3D reconstruction was performed using XMIPP 2.4 and EMAN1, CTFFIND3 for the CTF determination
CTF correctionDetails: CTFIND3, all micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: OTHER / Software - Name: XMIPP, 2.4, EMAN1 / Number images used: 20500
Final two d classificationNumber classes: 200

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