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- EMDB-34802: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubate... -

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Basic information

Entry
Database: EMDB / ID: EMD-34802
TitleOuter arm dynein of zebrafish sperm axoneme, calaxin-/-, incubated with recombinant Calaxin protein, refined focusing on the docking complex
Map dataouter arm dynein, calaxin-/- zebrafish sperm axoneme, incubated with recombinant Calaxin protein, refined focusing on the docking complex
Sample
  • Organelle or cellular component: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubated with recombinant Calaxin protein, refined focusing on the docking complex
Biological speciesDanio rerio (zebrafish)
Methodsubtomogram averaging / cryo EM / Resolution: 20.0 Å
AuthorsYamaguchi H / Kikkawa M
Funding support Japan, 3 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)16H02502 Japan
Japan Society for the Promotion of Science (JSPS)21H04762 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121002j001 Japan
CitationJournal: Elife / Year: 2023
Title: Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.
Authors: Hiroshi Yamaguchi / Motohiro Morikawa / Masahide Kikkawa /
Abstract: Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary ...Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of caused complete loss of OAD, whereas mutation of caused only partial loss of OAD. Detailed structural analysis revealed that OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates.
History
DepositionNov 16, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateApr 26, 2023-
Current statusApr 26, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34802.png

Downloads & links

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Map

FileDownload / File: emd_34802.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationouter arm dynein, calaxin-/- zebrafish sperm axoneme, incubated with recombinant Calaxin protein, refined focusing on the docking complex
Voxel sizeX=Y=Z: 6.14 Å
Density
Contour LevelBy AUTHOR: 1000.0
Minimum - Maximum-489.05493 - 2370.6282
Average (Standard dev.)743.0125 (±287.1799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 614.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34802_msk_1.map
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Additional map: masked

Fileemd_34802_additional_1.map
Annotationmasked
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Additional map: outer arm dynein, calaxin-/- zebrafish sperm axoneme, incubated...

Fileemd_34802_additional_2.map
Annotationouter arm dynein, calaxin-/- zebrafish sperm axoneme, incubated with recombinant Calaxin protein, 1 mM calcium condition, refined focusing on the docking complex
Projections & Slices
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Additional map: outer arm dynein, calaxin-/- zebrafish sperm axoneme, incubated...

Fileemd_34802_additional_3.map
Annotationouter arm dynein, calaxin-/- zebrafish sperm axoneme, incubated with recombinant mEGFP protein, refined focusing on the docking complex
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Additional map: outer arm dynein, calaxin-/- zebrafish sperm axoneme, incubated...

Fileemd_34802_additional_4.map
Annotationouter arm dynein, calaxin-/- zebrafish sperm axoneme, incubated with recombinant mEGFP-Calaxin protein, refined focusing on the docking complex
Projections & Slices
AxesZYX

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Half map: half map1, unmasked

Fileemd_34802_half_map_1.map
Annotationhalf map1, unmasked
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Half map: half map2, unmasked

Fileemd_34802_half_map_2.map
Annotationhalf map2, unmasked
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Sample components

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Entire : Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubate...

EntireName: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubated with recombinant Calaxin protein, refined focusing on the docking complex
Components
  • Organelle or cellular component: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubated with recombinant Calaxin protein, refined focusing on the docking complex

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Supramolecule #1: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubate...

SupramoleculeName: Outer arm dynein of zebrafish sperm axoneme, calaxin-/-, incubated with recombinant Calaxin protein, refined focusing on the docking complex
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Danio rerio (zebrafish) / Strain: TL / Organ: testis / Tissue: spermatozoa / Organelle: flagella / Location in cell: axoneme

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7.2
Details: 30 mM HEPES at pH 7.2, 5 mM MgSO4, 1 mM dithiothreitol, 1 mM EGTA, and 50 mM CH3COOK
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm / Nominal magnification: 15000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 35 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.45 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 9 / Number images used: 8480 / Software: (Name: IMOD (ver. 4.12.10), PEET (ver. 1.15.0))
Final angle assignmentType: NOT APPLICABLE / Software - Name: PEET (ver. 1.15.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET (ver. 1.15.0) / Number subtomograms used: 8468
FSC plot (resolution estimation)

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