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- EMDB-29666: 96-nm repeat unit of intact doublet microtubule from Tetrahymena ... -

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Basic information

Entry
Database: EMDB / ID: EMD-29666
Title96-nm repeat unit of intact doublet microtubule from Tetrahymena thermophila CFAP77AB-KO mutant
Map dataCFAP77ABKO 96 nm subtomogram average postprocess
Sample
  • Organelle or cellular component: 96-nm repeat unit of intact doublet microtubule from Tetrahymena thermophila CFAP77AB-KO mutant
KeywordsCilia / Axoneme / Doublet Microtubule / Microtubule Inner Protein / STRUCTURAL PROTEIN
Biological speciesTetrahymena thermophila (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 22.0 Å
AuthorsBlack CS / Legal T / Bui KH
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Commun / Year: 2023
Title: Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins.
Authors: Shintaroh Kubo / Corbin S Black / Ewa Joachimiak / Shun Kai Yang / Thibault Legal / Katya Peri / Ahmad Abdelzaher Zaki Khalifa / Avrin Ghanaeian / Caitlyn L McCafferty / Melissa Valente- ...Authors: Shintaroh Kubo / Corbin S Black / Ewa Joachimiak / Shun Kai Yang / Thibault Legal / Katya Peri / Ahmad Abdelzaher Zaki Khalifa / Avrin Ghanaeian / Caitlyn L McCafferty / Melissa Valente-Paterno / Chelsea De Bellis / Phuong M Huynh / Zhe Fan / Edward M Marcotte / Dorota Wloga / Khanh Huy Bui /
Abstract: Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine ...Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 42 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.
History
DepositionFeb 1, 2023-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29666.png

Downloads & links

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Map

FileDownload / File: emd_29666.map.gz / Format: CCP4 / Size: 18.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCFAP77ABKO 96 nm subtomogram average postprocess
Voxel sizeX=Y=Z: 8.48 Å
Density
Contour LevelBy AUTHOR: 18.0
Minimum - Maximum-18.422792000000001 - 57.919243000000002
Average (Standard dev.)2.069771 (±6.6644793)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions168168168
Spacing168168168
CellA=B=C: 1424.6399 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: CFAP77ABKO 96 nm subtomogram average half-map 1

Fileemd_29666_half_map_1.map
AnnotationCFAP77ABKO 96 nm subtomogram average half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CFAP77ABKO 96 nm subtomogram average half-map 2

Fileemd_29666_half_map_2.map
AnnotationCFAP77ABKO 96 nm subtomogram average half-map 2
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 repeat unit of intact doublet microtubule from Tetrahymena ...

EntireName: 96-nm repeat unit of intact doublet microtubule from Tetrahymena thermophila CFAP77AB-KO mutant
Components
  • Organelle or cellular component: 96-nm repeat unit of intact doublet microtubule from Tetrahymena thermophila CFAP77AB-KO mutant

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Supramolecule #1: 96-nm repeat unit of intact doublet microtubule from Tetrahymena ...

SupramoleculeName: 96-nm repeat unit of intact doublet microtubule from Tetrahymena thermophila CFAP77AB-KO mutant
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: Subtomogram average of the 96-nm repeating unit of the axoneme from intact cilia purified from CFAP77AB-KO mutant
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: CFAP77AB-KO / Organelle: Cilia

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

Concentration1.8 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 4.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 20 / Number images used: 1702
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 1702
FSC plot (resolution estimation)

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