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- EMDB-66326: Tomogram of doublet microtubules with bound dynein-2 molecules -

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Basic information

Entry
Database: EMDB / ID: EMD-66326
TitleTomogram of doublet microtubules with bound dynein-2 molecules
Map data
Sample
  • Complex: Complex of doublet microtubules and GST-Dyn2 molecules
    • Complex: SNAP-GST-Dyn2
    • Organelle or cellular component: Doublet microtubules
Keywordscilia / intraflagellar transport / doublet microtubule / dynein-2 / MOTOR PROTEIN
Biological speciesTetrahymena thermophila (eukaryote) / Homo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsHe HK / Chen X / Lv QH / Ichikawa M
Funding support China, Japan, 2 items
OrganizationGrant numberCountry
Other government24ZR1403800 China
Japan Science and TechnologyJPMJPR20E1 Japan
CitationJournal: EMBO J / Year: 2025
Title: Cryo-ET and MD simulations reveal that dynein-2 is tuned for binding to the A-tubule of the ciliary doublet.
Authors: Haoqiang K He / Shintaroh Kubo / Xuwei Chen / Qianru H Lv / Azusa Kage / Muneyoshi Ichikawa /
Abstract: Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along ...Eukaryotic cilia and flagella are thin structures present on the surface of cells, playing vital roles in signaling and cellular motion. Cilia assembly depends on intraflagellar transport (IFT) along doublet microtubules (doublets). Unlike dynein-1, which works on cytoplasmic singlet microtubules, dynein-2 works on the doublets inside cilia. Previous studies have shown that retrograde IFT, driven by dynein-2, occurs on the A-tubule of the doublet, suggesting an elusive mechanism by which dynein-2 recruits retrograde IFT to the A-tubule. Here, we investigated the molecular basis of this mechanism using cryo-electron tomography (cryo-ET), molecular dynamics (MD) simulations, and biochemical analysis. Our biochemical assays revealed that the microtubule-binding domain of dynein-2 exhibits a higher affinity for the ciliary doublets than dynein-1. Cryo-ET further visualized the preferential binding of dynein-2 to the A-tubule of the doublet. MD simulations suggest that dynein-2 prefers the tyrosinated tubulin lattice as is present in the A-tubule. These findings reveal a recruitment mechanism of retrograde IFT by dynein-2, providing new insights into the spatial and functional specialization of ciliary doublets.
History
DepositionSep 24, 2025-
Header (metadata) releaseDec 17, 2025-
Map releaseDec 17, 2025-
UpdateDec 17, 2025-
Current statusDec 17, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_66326.png

Downloads & links

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Map

FileDownload / File: emd_66326.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.53 Å/pix.
x 1366 pix.
= 11647.883 Å		8.53 Å/pix.
x 200 pix.
= 1705.4 Å		8.53 Å/pix.
x 1366 pix.
= 11647.883 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 8.527 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-18071.360000000000582 - 14108.575999999999112
Average (Standard dev.)2182.735799999999927 (±651.99676999999997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin30500
Dimensions20013661366
Spacing13662001366
CellA: 11647.883 Å / B: 1705.4001 Å / C: 11647.883 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Complex of doublet microtubules and GST-Dyn2 molecules

EntireName: Complex of doublet microtubules and GST-Dyn2 molecules
Components
  • Complex: Complex of doublet microtubules and GST-Dyn2 molecules
    • Complex: SNAP-GST-Dyn2
    • Organelle or cellular component: Doublet microtubules

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Supramolecule #1: Complex of doublet microtubules and GST-Dyn2 molecules

SupramoleculeName: Complex of doublet microtubules and GST-Dyn2 molecules
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Location in cell: Cilia

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Supramolecule #2: SNAP-GST-Dyn2

SupramoleculeName: SNAP-GST-Dyn2 / type: complex / ID: 2 / Parent: 1
Details: Dynein-2 heavy chain's N-terminal tail domain was truncated and replaced with GST-tag for dimerization. There is also SNAP-tag attached at the N-terminal side.
Source (natural)Organism: Homo sapiens (human) / Location in cell: Cilia

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Supramolecule #3: Doublet microtubules

SupramoleculeName: Doublet microtubules / type: organelle_or_cellular_component / ID: 3 / Parent: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Organelle: Cilia

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statefilament

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Sample preparation

BufferpH: 6.9
GridModel: Quantifoil R2/2 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Sigma-Aldrich / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number real images: 26 / Average electron dose: 3.065 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: IMOD / Number images used: 26
CTF correctionType: NONE

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