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- EMDB-6289: Three dimensional reconstruction of bovine dynactin complex by cr... -

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Basic information

Entry
Database: EMDB / ID: EMD-6289
TitleThree dimensional reconstruction of bovine dynactin complex by cryo electron microscopy
Map dataThree dimensional reconstruction of bovine dynactin complex by cryo electron microscopy
Sample
  • Sample: Three-dimensional reconstruction of bovine dynactin complex by cryo EM
  • Protein or peptide: Arp1
  • Protein or peptide: Actin
  • Protein or peptide: Arp11
  • Protein or peptide: p62
  • Protein or peptide: p25
  • Protein or peptide: p27
  • Protein or peptide: CapZ alpha
  • Protein or peptide: CapZ beta
Keywordsdynactin / actin-related proteins / dynein
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsChowdhury S / Ketcham SA / Schroer TA / Lander GC
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: Structural organization of the dynein-dynactin complex bound to microtubules.
Authors: Saikat Chowdhury / Stephanie A Ketcham / Trina A Schroer / Gabriel C Lander /
Abstract: Cytoplasmic dynein associates with dynactin to drive cargo movement on microtubules, but the structure of the dynein-dynactin complex is unknown. Using electron microscopy, we determined the ...Cytoplasmic dynein associates with dynactin to drive cargo movement on microtubules, but the structure of the dynein-dynactin complex is unknown. Using electron microscopy, we determined the organization of native bovine dynein, dynactin and the dynein-dynactin-microtubule quaternary complex. In the microtubule-bound complex, the dynein motor domains are positioned for processive unidirectional movement, and the cargo-binding domains of both dynein and dynactin are accessible.
History
DepositionFeb 27, 2015-
Header (metadata) releaseMar 11, 2015-
Map releaseMar 11, 2015-
UpdateMay 27, 2015-
Current statusMay 27, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.69
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.69
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6289.png

Downloads & links

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Map

FileDownload / File: emd_6289.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree dimensional reconstruction of bovine dynactin complex by cryo electron microscopy
Voxel sizeX=Y=Z: 2.62 Å
Density
Contour LevelBy AUTHOR: 3.69 / Movie #1: 3.69
Minimum - Maximum-30.655632019999999 - 45.313503269999998
Average (Standard dev.)0.00019436 (±0.95106626)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions258258258
Spacing258258258
CellA=B=C: 675.95996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z258258258
origin x/y/z0.0000.0000.000
length x/y/z675.960675.960675.960
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS258258258
D min/max/mean-30.65645.3140.000

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Supplemental data

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Sample components

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Entire : Three-dimensional reconstruction of bovine dynactin complex by cryo EM

EntireName: Three-dimensional reconstruction of bovine dynactin complex by cryo EM
Components
  • Sample: Three-dimensional reconstruction of bovine dynactin complex by cryo EM
  • Protein or peptide: Arp1
  • Protein or peptide: Actin
  • Protein or peptide: Arp11
  • Protein or peptide: p62
  • Protein or peptide: p25
  • Protein or peptide: p27
  • Protein or peptide: CapZ alpha
  • Protein or peptide: CapZ beta

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Supramolecule #1000: Three-dimensional reconstruction of bovine dynactin complex by cryo EM

SupramoleculeName: Three-dimensional reconstruction of bovine dynactin complex by cryo EM
type: sample / ID: 1000
Details: The sample was monodisperse. The shoulder domain of dynactin would dissociate during cryo grid preparation.
Oligomeric state: One CapZ alpha, one capZ beta, eight Arp1, one actin, one Arp11, one p25, one p27, one p62
Number unique components: 8
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: Arp1

MacromoleculeName: Arp1 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Oligomeric state: Octamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 42.6 KDa

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Macromolecule #2: Actin

MacromoleculeName: Actin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 42 KDa

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Macromolecule #3: Arp11

MacromoleculeName: Arp11 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 42 KDa

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Macromolecule #4: p62

MacromoleculeName: p62 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 62 KDa

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Macromolecule #5: p25

MacromoleculeName: p25 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 25 KDa

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Macromolecule #6: p27

MacromoleculeName: p27 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 27 KDa

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Macromolecule #7: CapZ alpha

MacromoleculeName: CapZ alpha / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 33 KDa

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Macromolecule #8: CapZ beta

MacromoleculeName: CapZ beta / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: Brain / Cell: Neuron / Location in cell: Cytoplasm
Molecular weightTheoretical: 33 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

Concentration0.05 mg/mL
BufferpH: 7.2 / Details: 35 mM Tris, 5 mM MgSO4, 150 mM KCl, 1 mM TCEP
GridDetails: 400 mesh C-flat holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 83.15 K / Instrument: HOMEMADE PLUNGER
Details: External environment at 4 degrees Celsius, 98% humidity.
Method: 4 uL dynactin sample was applied to a cflat holey grid. Excess sample was manually blotted using filter paper for 5-7 seconds and immediately vitrified by plunge-freezing into liquid ethane ...Method: 4 uL dynactin sample was applied to a cflat holey grid. Excess sample was manually blotted using filter paper for 5-7 seconds and immediately vitrified by plunge-freezing into liquid ethane slurry at -179 degrees Celsius.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 22500
Sample stageSpecimen holder: Liquid nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 83 K / Max: 85 K / Average: 84 K
Alignment procedureLegacy - Astigmatism: Objective astigmatism was corrected using a quadrupole stigmator at 22,500 times magnification.
DetailsImaging was done on a Gatan K2 Summit camera operated in counting mode at a dose rate of 10 electrons/pixel/s. Each movie comprised 30 frames acquired over 6s, with a cumulative dose of 35 electrons/A**2.
DateOct 20, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2421 / Average electron dose: 35 e/Å2
Details: Data collected using Leginon automated image acquisition software. Each movie comprised 30 frames acquired over 6 seconds.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final angle assignmentDetails: Theta 45 degrees, phi 45 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: OTHER / Software - Name: Relion
Details: Processing leading up to 3D reconstruction was performed using the Appion package. Particles were selected from micrographs using a template-based automated particle picker. The stack was ...Details: Processing leading up to 3D reconstruction was performed using the Appion package. Particles were selected from micrographs using a template-based automated particle picker. The stack was subjected to five iterations of iterative 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). The clean particle stack was subjected to 25 iterations of 3D classification with three classes using the Relion suite. Particles belonging to well-resolved 3D class averages were used for further refinement by projection matching in Relion.
Number images used: 59538
DetailsProcessing leading up to 3D reconstruction was performed using the Appion package. Particles were selected from micrographs using a template-based automated particle picker. The stack was subjected to five iterations of iterative 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). The clean particle stack was subjected to 25 iterations of 3D classification with three classes using the Relion suite. Particles belonging to well-resolved 3D class averages were used for further refinement by projection matching in Relion.

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