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- EMDB-14723: Tomogram of bovine sperm singlet microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-14723
TitleTomogram of bovine sperm singlet microtubules
Map dataTomographic reconstruction of a bovine sperm flagellum tip. Striations of TAILS are visible along the microtubules. Reconstructed with IMOD package.
Sample
  • Organelle or cellular component: Bovine sperm flagellum tip
KeywordsFlagella Cilia Microtubules Sperm / UNKNOWN FUNCTION
Biological speciesBos taurus (cattle)
Methodelectron tomography / cryo EM
AuthorsHoog JL / Zabeo D / Croft JT
Funding support Sweden, 2 items
OrganizationGrant numberCountry
Swedish Research Council2015-05427 Sweden
Swedish Research Council2019-04004 Sweden
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules.
Authors: Miao Gui / Jacob T Croft / Davide Zabeo / Vajradhar Acharya / Justin M Kollman / Thomas Burgoyne / Johanna L Höög / Alan Brown /
Abstract: The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins ...The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.
History
DepositionApr 5, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14723.png

Downloads & links

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Map

FileDownload / File: emd_14723.map.gz / Format: CCP4 / Size: 3.3 GB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationTomographic reconstruction of a bovine sperm flagellum tip. Striations of TAILS are visible along the microtubules. Reconstructed with IMOD package.
Voxel sizeX=Y=Z: 4.37 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)0.21419631 (±24.428668999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-73-6543
Dimensions33723296321
Spacing32963372321
CellA: 14403.52 Å / B: 14735.64 Å / C: 1402.77 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Bovine sperm flagellum tip

EntireName: Bovine sperm flagellum tip
Components
  • Organelle or cellular component: Bovine sperm flagellum tip

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Supramolecule #1: Bovine sperm flagellum tip

SupramoleculeName: Bovine sperm flagellum tip / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Bos taurus (cattle) / Tissue: Sperm / Organelle: Flagellum / Location in cell: Flagellum end piece

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K
DetailsThe sperm cells were washed once in 1x PBS at 300x g for 8 minutes.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average exposure time: 3.5 sec. / Average electron dose: 1.64 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 33000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Number images used: 61

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