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- EMDB-2716: Dynactin 3D structure: Implications for assembly and dynein binding -

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Basic information

Entry
Database: EMDB / ID: EMD-2716
TitleDynactin 3D structure: Implications for assembly and dynein binding
Map dataRandom conical tilt 3D reconstruction of negatively stained dynactin complex
Sample
  • Sample: The dynactin complex
  • Protein or peptide: p150Glued
  • Protein or peptide: p50/dynamitin
  • Protein or peptide: p24
  • Protein or peptide: Arp1
  • Protein or peptide: Arp11
  • Protein or peptide: actin
  • Protein or peptide: p25
  • Protein or peptide: p27
  • Protein or peptide: p62
Keywordsdynein / microtubule / actin / single particle analysis
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / negative staining / Resolution: 34.0 Å
AuthorsImai H / Narita A / Maeda Y / Schroer TA
CitationJournal: J Mol Biol / Year: 2014
Title: Dynactin 3D structure: implications for assembly and dynein binding.
Authors: Hiroshi Imai / Akihiro Narita / Yuichiro Maéda / Trina A Schroer /
Abstract: The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been ...The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either ≈1MDa assembly. In the present study, we used negative-stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional (3D) structure of native vertebrate dynactin. The 35-nm-long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other end, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in two-dimensional images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7 Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.
History
DepositionJul 24, 2014-
Header (metadata) releaseAug 6, 2014-
Map releaseAug 13, 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: 0.001968
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.001968
  • 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_2716.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRandom conical tilt 3D reconstruction of negatively stained dynactin complex
Voxel sizeX=Y=Z: 3.5 Å
Density
Contour LevelBy AUTHOR: 0.001968 / Movie #1: 0.001968
Minimum - Maximum-0.00096521 - 0.01285007
Average (Standard dev.)0.00016198 (±0.00095364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions8080160
Spacing8080160
CellA: 280.0 Å / B: 280.0 Å / C: 560.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.53.53.5
M x/y/z8080160
origin x/y/z0.0000.0000.000
length x/y/z280.000280.000560.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ442626
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS8080160
D min/max/mean-0.0010.0130.000

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Supplemental data

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Sample components

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Entire : The dynactin complex

EntireName: The dynactin complex
Components
  • Sample: The dynactin complex
  • Protein or peptide: p150Glued
  • Protein or peptide: p50/dynamitin
  • Protein or peptide: p24
  • Protein or peptide: Arp1
  • Protein or peptide: Arp11
  • Protein or peptide: actin
  • Protein or peptide: p25
  • Protein or peptide: p27
  • Protein or peptide: p62

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Supramolecule #1000: The dynactin complex

SupramoleculeName: The dynactin complex / type: sample / ID: 1000
Details: Experimental weight (MDa) 1.11 + 0.12 megadaltons (mean+SD; n=151) scanning transmission electron microscopy (STEM) analysis (Schafer et al., 1994 J. Cell Biol.126: 403-412.) Theoretical ...Details: Experimental weight (MDa) 1.11 + 0.12 megadaltons (mean+SD; n=151) scanning transmission electron microscopy (STEM) analysis (Schafer et al., 1994 J. Cell Biol.126: 403-412.) Theoretical weight (MDa) 0.95-1.04 megadaltons. (see table I of Imai et al., 2014 J. Mol. Biol.)
Oligomeric state: Two p150Glued, Four p50/dynamitin, two p24, five, six or seven Arp1, one actin, one Arp11, one p25, one p27, one p62
Number unique components: 9

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Macromolecule #1: p150Glued

MacromoleculeName: p150Glued / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #2: p50/dynamitin

MacromoleculeName: p50/dynamitin / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #3: p24

MacromoleculeName: p24 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #4: Arp1

MacromoleculeName: Arp1 / type: protein_or_peptide / ID: 4 / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #5: Arp11

MacromoleculeName: Arp11 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #6: actin

MacromoleculeName: actin / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #7: p25

MacromoleculeName: p25 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #8: p27

MacromoleculeName: p27 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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Macromolecule #9: p62

MacromoleculeName: p62 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Gallus gallus (chicken) / Tissue: Embryo Brain

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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

Concentration0.015 mg/mL
BufferpH: 7.1
Details: 35 mM Pipes-KOH, 5 mM MgSO4, 1 mM EGTA, and 0.5 mM EDTA, ~30 mM KCl
StainingType: NEGATIVE
Details: Dynactin complex was negatively stained with 1% uranyl acetate on glow-discharged carbon grids.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2010HC
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 20000
Sample stageSpecimen holder: room temperature, high tilt holder / Specimen holder model: JEOL / Tilt angle max: 60
DateJan 19, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Average electron dose: 18 e/Å2
Tilt angle min0

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 34.0 Å / Resolution method: OTHER / Software - Name: Eos, SPIDER
Details: Images of the dynactin structure were divided into two groups and reconstructed into two 3D maps that were compared by Fourier Shell Correlation (Van Heel, 1987).
Number images used: 3637
DetailsRandom conical tilt reconstruction

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