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- EMDB-2857: CryoEM reconstruction of dynactin complex from pig brain at 4.0 A... -

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Entry
Database: EMDB / ID: EMD-2857
TitleCryoEM reconstruction of dynactin complex from pig brain at 4.0 Angstrom resolution overall
Map dataCryoEM reconstruction of the Arp filament of dynactin complex from at 3.5 angstrom resolution
Sample
  • Sample: Dynactin complex from pig brainDynactin
  • Protein or peptide: Actin related protein 1
  • Protein or peptide: Actin related protein 11
  • Protein or peptide: beta-actin
  • Protein or peptide: Dynactin subunit 1
  • Protein or peptide: Dynactin subunit 2
  • Protein or peptide: Dynactin subunit 3
  • Protein or peptide: Actin capping protein
  • Protein or peptide: Dynactin subunit 4
  • Protein or peptide: Dynactin subunit 5
  • Protein or peptide: Dynactin subunit 6
Keywordsdynactin / dynein co-factor / actin-like filament / cellular cargo transport
Function / homology
Function and homology information


retrograde axonal transport of mitochondrion / Gap junction degradation / Formation of annular gap junctions / Regulation of actin dynamics for phagocytic cup formation / EPHB-mediated forward signaling / VEGFA-VEGFR2 Pathway / Cell-extracellular matrix interactions / RHO GTPases Activate WASPs and WAVEs / MAP2K and MAPK activation / dynactin complex ...retrograde axonal transport of mitochondrion / Gap junction degradation / Formation of annular gap junctions / Regulation of actin dynamics for phagocytic cup formation / EPHB-mediated forward signaling / VEGFA-VEGFR2 Pathway / Cell-extracellular matrix interactions / RHO GTPases Activate WASPs and WAVEs / MAP2K and MAPK activation / dynactin complex / Clathrin-mediated endocytosis / F-actin capping protein complex / cellular response to cytochalasin B / regulation of transepithelial transport / structural constituent of postsynaptic actin cytoskeleton / morphogenesis of a polarized epithelium / postsynaptic actin cytoskeleton / protein localization to adherens junction / dense body / Tat protein binding / Neutrophil degranulation / dynein complex / apical protein localization / barbed-end actin filament capping / adherens junction assembly / coronary vasculature development / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / tight junction / regulation of norepinephrine uptake / aorta development / COPI-mediated anterograde transport / NuA4 histone acetyltransferase complex / regulation of synaptic vesicle endocytosis / apical junction complex / ventricular septum development / establishment or maintenance of cell polarity / dynein complex binding / cortical cytoskeleton / positive regulation of double-strand break repair via homologous recombination / nitric-oxide synthase binding / brush border / kinesin binding / calyx of Held / regulation of protein localization to plasma membrane / microtubule-based process / axon cytoplasm / axonogenesis / mitotic spindle organization / cell motility / actin filament / adherens junction / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Schaffer collateral - CA1 synapse / kinetochore / cytoplasmic ribonucleoprotein granule / nucleosome / actin cytoskeleton / lamellipodium / actin binding / cell cortex / actin cytoskeleton organization / nuclear membrane / cytoskeleton / hydrolase activity / regulation of cell cycle / ribonucleoprotein complex / axon / focal adhesion / centrosome / glutamatergic synapse / synapse / protein kinase binding / protein-containing complex / nucleoplasm / ATP binding / membrane / nucleus / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
: / Dynamitin / Dynamitin / Dynactin subunit 6 / F-actin-capping protein subunit beta / F-actin capping protein, beta subunit, conserved site / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit / F-actin capping protein beta subunit signature. / F-actin capping protein, alpha subunit, conserved site ...: / Dynamitin / Dynamitin / Dynactin subunit 6 / F-actin-capping protein subunit beta / F-actin capping protein, beta subunit, conserved site / F-actin-capping protein subunit beta, N-terminal domain / F-actin capping protein, beta subunit / F-actin capping protein beta subunit signature. / F-actin capping protein, alpha subunit, conserved site / F-actin capping protein alpha subunit signature 1. / F-actin capping protein alpha subunit signature 2. / F-actin-capping protein subunit alpha / F-actin-capping protein subunit alpha/beta / F-actin-capping protein subunit alpha/beta, domain 2 / F-actin capping protein, alpha subunit, domain 1 / F-actin capping protein alpha subunit / Trimeric LpxA-like superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Dynactin subunit 5 / Dynactin / Dynactin subunit 2 / F-actin-capping protein subunit alpha-1 / Dynactin subunit 6 / F-actin-capping protein subunit beta / Alpha-centractin / Actin-related protein 10 / Actin, cytoplasmic 1
Similarity search - Component
Biological speciesSus scrofa domesticus (domestic pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang K / Urnavicius L / Diamant AG / Motz C / Schlager MA / Yu M / Patel NA / Robinson CV / Carter AP
CitationJournal: Science / Year: 2015
Title: The structure of the dynactin complex and its interaction with dynein.
Authors: Linas Urnavicius / Kai Zhang / Aristides G Diamant / Carina Motz / Max A Schlager / Minmin Yu / Nisha A Patel / Carol V Robinson / Andrew P Carter /
Abstract: Dynactin is an essential cofactor for the microtubule motor cytoplasmic dynein-1. We report the structure of the 23-subunit dynactin complex by cryo-electron microscopy to 4.0 angstroms. Our ...Dynactin is an essential cofactor for the microtubule motor cytoplasmic dynein-1. We report the structure of the 23-subunit dynactin complex by cryo-electron microscopy to 4.0 angstroms. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin-related protein Arp1 and one of β-actin. The filament is capped at each end by distinct protein complexes, and its length is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2 angstrom structure of the complex between dynein, dynactin, and the motility-inducing cargo adaptor Bicaudal-D2 shows how the translational symmetry of the dynein tail matches that of the dynactin filament. The Bicaudal-D2 coiled coil runs between dynein and dynactin to stabilize the mutually dependent interactions between all three components.
History
DepositionJan 21, 2015-
Header (metadata) releaseMar 4, 2015-
Map releaseMar 4, 2015-
UpdateApr 6, 2016-
Current statusApr 6, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.168
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.168
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5adx
  • Surface level: 0.168
  • 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_2857.map.gz / Format: CCP4 / Size: 16.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM reconstruction of the Arp filament of dynactin complex from at 3.5 angstrom resolution
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.133 / Movie #1: 0.168
Minimum - Maximum-0.30919424 - 0.64745843
Average (Standard dev.)0.00548598 (±0.0328186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-62-94-95
Dimensions111211189
Spacing111211189
CellA: 282.74002 Å / B: 148.74 Å / C: 253.26001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z211111189
origin x/y/z0.0000.0000.000
length x/y/z282.740148.740253.260
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-94-62-95
NC/NR/NS211111189
D min/max/mean-0.3090.6470.005

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Supplemental data

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Sample components

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Entire : Dynactin complex from pig brain

EntireName: Dynactin complex from pig brainDynactin
Components
  • Sample: Dynactin complex from pig brainDynactin
  • Protein or peptide: Actin related protein 1
  • Protein or peptide: Actin related protein 11
  • Protein or peptide: beta-actin
  • Protein or peptide: Dynactin subunit 1
  • Protein or peptide: Dynactin subunit 2
  • Protein or peptide: Dynactin subunit 3
  • Protein or peptide: Actin capping protein
  • Protein or peptide: Dynactin subunit 4
  • Protein or peptide: Dynactin subunit 5
  • Protein or peptide: Dynactin subunit 6

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Supramolecule #1000: Dynactin complex from pig brain

SupramoleculeName: Dynactin complex from pig brain / type: sample / ID: 1000
Details: he sample was stored in -80 degrees Celcius freezer before being loaded onto the grid.
Oligomeric state: One dynactin complex / Number unique components: 10
Molecular weightExperimental: 1.06 MDa / Theoretical: 1.06 MDa / Method: Mass spectrometry

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Macromolecule #1: Actin related protein 1

MacromoleculeName: Actin related protein 1 / type: protein_or_peptide / ID: 1 / Name.synonym: Arp1 / Number of copies: 8 / Oligomeric state: Octamer in actin like filament / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 42.6 KDa / Theoretical: 42.6 KDa

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Macromolecule #2: Actin related protein 11

MacromoleculeName: Actin related protein 11 / type: protein_or_peptide / ID: 2 / Name.synonym: Arp11 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 46.3 KDa / Theoretical: 46.3 KDa

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Macromolecule #3: beta-actin

MacromoleculeName: beta-actin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 43 KDa / Theoretical: 43 KDa

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Macromolecule #4: Dynactin subunit 1

MacromoleculeName: Dynactin subunit 1 / type: protein_or_peptide / ID: 4 / Name.synonym: DCTN1 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 150 KDa / Theoretical: 150 KDa

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Macromolecule #5: Dynactin subunit 2

MacromoleculeName: Dynactin subunit 2 / type: protein_or_peptide / ID: 5 / Name.synonym: DCTN2 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa

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Macromolecule #6: Dynactin subunit 3

MacromoleculeName: Dynactin subunit 3 / type: protein_or_peptide / ID: 6 / Name.synonym: DCTN3 / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 22 KDa / Theoretical: 22 KDa

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Macromolecule #7: Actin capping protein

MacromoleculeName: Actin capping protein / type: protein_or_peptide / ID: 7 / Name.synonym: CapZ / Number of copies: 2 / Oligomeric state: Heterodimer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 33 KDa / Theoretical: 33 KDa

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Macromolecule #8: Dynactin subunit 4

MacromoleculeName: Dynactin subunit 4 / type: protein_or_peptide / ID: 8 / Name.synonym: DCTN4 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 62 KDa / Theoretical: 62 KDa

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Macromolecule #9: Dynactin subunit 5

MacromoleculeName: Dynactin subunit 5 / type: protein_or_peptide / ID: 9 / Name.synonym: DCTN5 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa

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Macromolecule #10: Dynactin subunit 6

MacromoleculeName: Dynactin subunit 6 / type: protein_or_peptide / ID: 10 / Name.synonym: DCTN6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / synonym: Pig / Tissue: Brain / Location in cell: Cytoplasm
Molecular weightExperimental: 27 KDa / Theoretical: 27 KDa

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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

Concentration0.07 mg/mL
BufferpH: 6.5
Details: 50mM KCl, 25mM KH2PO4-K2HPO4, 5mM DDT, 1mM MgCl2, 0.1 mM ATP
GridDetails: R2/2 400 square mesh copper grid with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 105 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 102967 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 96,000 times nominal magnification
Legacy - Electron beam tilt params: 0
DateSep 12, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 14423 / Average electron dose: 54 e/Å2 / Bits/pixel: 32
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle by Gctf
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: OTHER / Software - Name: Relion
Details: Final map were sharpened to 3.5 angstrom from a 4.1 angstrom map in overall This map was only used for analysis of Arp filament of dynactin
Number images used: 115044
DetailsThe particles were selected using a GPU accelerated automatic program Gautomatch. The CTF parameter were determined and refined using a GPU accelerated program Gctf.

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