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- EMDB-45870: Ciliary 96-nm repeat unit from active and EHNA-inhibited sperm ce... -

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Basic information

Entry
Database: EMDB / ID: EMD-45870
TitleCiliary 96-nm repeat unit from active and EHNA-inhibited sperm cells of the American eel (Anguilla rostrata)
Map dataSubtomogram average of the 96-nm repeating axonemal unit from active and EHNA-inhibited eel sperm flagella.
Sample
  • Organelle or cellular component: 96-nm axoneme repeat unit from active and EHNA-inhbited American eel sperm flagella
Keywordscilia / flagella / axoneme / MOTOR PROTEIN
Biological speciesAnguilla rostrata (American eel)
Methodsubtomogram averaging / cryo EM / Resolution: 20.0 Å
AuthorsSchrad JR / Fu G / Hable WE / Tayar AM / Oliveira K / Nicastro D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM083122 United States
Life Sciences Research Foundation United States
CitationJournal: Mol Biol Cell / Year: 2025
Title: Cryo-electron tomography of eel sperm flagella reveals a molecular "minimum system" for motile cilia.
Authors: Jason R Schrad / Gang Fu / Whitney E Hable / Alexandra M Tayar / Kenneth Oliveira / Daniela Nicastro /
Abstract: Cilia and flagella play a crucial role in the development and function of eukaryotes. The activity of thousands of dyneins is precisely regulated to generate flagellar motility. The complex proteome ...Cilia and flagella play a crucial role in the development and function of eukaryotes. The activity of thousands of dyneins is precisely regulated to generate flagellar motility. The complex proteome (600+ proteins) and architecture of the structural core of flagella, the axoneme, have made it challenging to dissect the functions of the different complexes, like the regulatory machinery. Previous reports suggested that the flagellum of American eel sperm lacks many of the canonical axonemal complexes yet is still motile. Here, we use cryo-electron tomography for molecular characterization of this proposed "minimal" motile flagellum. We observed different diameters for the eel sperm flagellum: narrow at the base and wider toward the flagellar tip. Subtomogram averaging revealed the three-dimensional (3D) structure of the eel sperm flagellum. As expected, major complexes were missing, for example, outer dynein arms, radial spokes, and the central pair complex, but we found molecular remnants of most complexes. We also identified bend direction-specific patterns in the inter-DMT distance in actively beating eel sperm flagella and we propose a model for the regulation of dynein activity during their motility. Together, our results shed light on the structure and function of the eel sperm flagellum and provide insight into the minimum requirements for ciliary beating.
History
DepositionJul 22, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 5, 2025-
Current statusMar 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45870.png

Downloads & links

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Map

FileDownload / File: emd_45870.map.gz / Format: CCP4 / Size: 54.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the 96-nm repeating axonemal unit from active and EHNA-inhibited eel sperm flagella.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.15 Å/pix.
x 232 pix.
= 731.032 Å		3.15 Å/pix.
x 196 pix.
= 617.596 Å		3.15 Å/pix.
x 315 pix.
= 992.565 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 3.151 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 126.5
Minimum - Maximum112.267870000000002 - 139.028140000000008
Average (Standard dev.)125.504450000000006 (±3.31425)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-118-133-80
Dimensions196315232
Spacing315196232
CellA: 992.565 Å / B: 617.596 Å / C: 731.032 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half maps are unwindowed, they need to be...

Fileemd_45870_half_map_1.map
AnnotationHalf maps are unwindowed, they need to be windowed, tapered, masked or etc. before FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half maps are unwindowed, they need to be...

Fileemd_45870_half_map_2.map
AnnotationHalf maps are unwindowed, they need to be windowed, tapered, masked or etc. before FSC calculation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 96-nm axoneme repeat unit from active and EHNA-inhbited American ...

EntireName: 96-nm axoneme repeat unit from active and EHNA-inhbited American eel sperm flagella
Components
  • Organelle or cellular component: 96-nm axoneme repeat unit from active and EHNA-inhbited American eel sperm flagella

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Supramolecule #1: 96-nm axoneme repeat unit from active and EHNA-inhbited American ...

SupramoleculeName: 96-nm axoneme repeat unit from active and EHNA-inhbited American eel sperm flagella
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Anguilla rostrata (American eel) / Tissue: Sperm cells / Organelle: Flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
360.0 mMNaClsodium chloride
50.0 mMMgCl2magnesium chloride
10.0 mMCaCl2calcium chloride
10.0 mMKClpotassium chloride
30.0 mMHEPES

Details: Artificial sea water, used for sperm cell activation
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Back-side blotting for 3-5 seconds before plunging..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 26000
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 reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software: (Name: IMOD, PEET) / Number subtomograms used: 3832
ExtractionNumber tomograms: 32 / Number images used: 3832 / Software: (Name: IMOD, PEET)
Final angle assignmentType: NOT APPLICABLE

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