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- EMDB-7001: Subtomogram average of two dynein tail domains bound to dynactin-... -

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Basic information

Entry
Database: EMDB / ID: EMD-7001
TitleSubtomogram average of two dynein tail domains bound to dynactin-Hook3 complex
Map datadynein tail domains associated with dynactin-Hook3
Sample
  • Complex: two dynein tail domains bound to dynactin-Hook3 complex
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 38.0 Å
AuthorsGrotjahn DA / Chowdhury S / Xu Y / McKenney RJ / Schroer TA / Lander GC
CitationJournal: Nat Struct Mol Biol / Year: 2018
Title: Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility.
Authors: Danielle A Grotjahn / Saikat Chowdhury / Yiru Xu / Richard J McKenney / Trina A Schroer / Gabriel C Lander /
Abstract: Cytoplasmic dynein is a protein complex that transports molecular cargo along microtubules (MTs), playing a key role in the intracellular trafficking network. Vertebrate dynein's movement becomes ...Cytoplasmic dynein is a protein complex that transports molecular cargo along microtubules (MTs), playing a key role in the intracellular trafficking network. Vertebrate dynein's movement becomes strikingly enhanced upon interacting with dynactin and a cargo adaptor such as BicaudalD2. However, the mechanisms responsible for increased transport activity are not well understood, largely owing to limited structural information. We used cryo-electron tomography (cryo-ET) to visualize the 3D structure of the MT-bound dynein-dynactin complex from Mus musculus and show that the dynactin-cargo adaptor complex binds two dimeric dyneins. This configuration imposes spatial and conformational constraints on both dynein dimers, positioning the four motor domains in proximity to one another and oriented toward the MT minus end. We propose that grouping multiple dyneins onto a single dynactin scaffold promotes collective force production, increased processivity, and unidirectional movement, suggesting mechanistic parallels to axonemal dynein. These findings provide structural insights into a previously unknown mechanism for dynein regulation.
History
DepositionAug 23, 2017-
Header (metadata) releaseSep 20, 2017-
Map releaseSep 20, 2017-
UpdateMar 21, 2018-
Current statusMar 21, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.14
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.14
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7001.png

Downloads & links

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Map

FileDownload / File: emd_7001.map.gz / Format: CCP4 / Size: 2.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdynein tail domains associated with dynactin-Hook3
Voxel sizeX=Y=Z: 8.52 Å
Density
Contour LevelBy AUTHOR: 0.14 / Movie #1: 0.14
Minimum - Maximum-0.63707274 - 1.0863092
Average (Standard dev.)-0.00014936713 (±0.04919312)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions848484
Spacing848484
CellA=B=C: 715.68005 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.528.528.52
M x/y/z848484
origin x/y/z0.0000.0000.000
length x/y/z715.680715.680715.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS848484
D min/max/mean-0.6371.086-0.000

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Supplemental data

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Sample components

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Entire : two dynein tail domains bound to dynactin-Hook3 complex

EntireName: two dynein tail domains bound to dynactin-Hook3 complex
Components
  • Complex: two dynein tail domains bound to dynactin-Hook3 complex

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Supramolecule #1: two dynein tail domains bound to dynactin-Hook3 complex

SupramoleculeName: two dynein tail domains bound to dynactin-Hook3 complex
type: complex / ID: 1 / Parent: 0
Details: SNAPf-Hook3 fragment recombinantly expressed and purified. Microtubule-bound dynein-dynactin complexes isolated from mouse brain tissue.
Source (natural)Organism: Mus musculus (house mouse) / Strain: C57BL/6J
Molecular weightTheoretical: 4.2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
Details: PMEE buffer (35 mM PIPES, 5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, 1 mM GTP, 4 mM AMPPNP, 20 uM Taxol)
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 4 uL of sample was applied to one side of a plasma-cleaned grid, and the grid was manually blotted on the opposite side for 5-7 seconds immediately before vitrification..
DetailsMicrotubule-bound dynein-dynactin-Hook3 complexes isolated from mouse brain tissue in the presence of non-hydrolyzable ATP analog AMPPNP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 14000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 14000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 98.15 K / Max: 98.15 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-15 / Number grids imaged: 1 / Average exposure time: 1.16 sec. / Average electron dose: 0.95 e/Å2
Details: Images were collected in movie mode with a per-frame exposure time of 80ms.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 13 / Number images used: 303 / Method: manual / Software - Name: EMAN2 (ver. 2.11) / Software - details: e2spt_boxer
Details: Subvolumes were picked using the EMAN2 manual picker for subvolumes.
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4) / Software - details: Subtomogram Averaging
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 38.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 1.4)
Details: Initial angular assignments were obtained by manually docking available structures/reconstructions into extracted subvolumes using UCSF Chimera. These were further refined using the RELION 1. ...Details: Initial angular assignments were obtained by manually docking available structures/reconstructions into extracted subvolumes using UCSF Chimera. These were further refined using the RELION 1.4 subtomogram averaging auto-refinement program, followed by focused refinement of individual subregions of the complex by application of binary masks and continuation of refinement. The resulting focused maps were stitched together using the "vop maximum" function in UCSF Chimera.
Number subtomograms used: 502
DetailsMovie frames per tilt were aligned using MotionCorr.

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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