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- EMDB-18124: A cryo-ET study of ciliary rootlet organization - purified rootle... -

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Basic information

Entry
Database: EMDB / ID: EMD-18124
TitleA cryo-ET study of ciliary rootlet organization - purified rootlet example 3
Map datapurified rootlet, eman2 preprocessed cropped tomogram for segmentation. Object files are found on biostudies (S-BSST1164, https://www.ebi.ac.uk/biostudies/studies/S-BSST1164)
Sample
  • Organelle or cellular component: Ciliary rootlet surrounded by cellular membranes
Keywordsciliary rootlet / rootlet / rootletin / centriole / cilia / STRUCTURAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
Authorsvan Hoorn C / Carter AP
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1011 United Kingdom
Wellcome Trust210711/Z/18/Z United Kingdom
CitationJournal: Elife / Year: 2024
Title: A cryo-electron tomography study of ciliary rootlet organization.
Authors: Chris van Hoorn / Andrew P Carter /
Abstract: Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the ...Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet's filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.
History
DepositionAug 3, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: PDBe / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_18124.map.gz / Format: CCP4 / Size: 73.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpurified rootlet, eman2 preprocessed cropped tomogram for segmentation. Object files are found on biostudies (S-BSST1164, https://www.ebi.ac.uk/biostudies/studies/S-BSST1164)
Voxel sizeX=Y=Z: 11.104 Å
Density
Minimum - Maximum-3.0 - 3.0
Average (Standard dev.)0.01585852 (±0.92345303)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin170153140
Dimensions48041696
Spacing41648096
CellA: 4619.264 Å / B: 5329.92 Å / C: 1065.984 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ciliary rootlet surrounded by cellular membranes

EntireName: Ciliary rootlet surrounded by cellular membranes
Components
  • Organelle or cellular component: Ciliary rootlet surrounded by cellular membranes

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Supramolecule #1: Ciliary rootlet surrounded by cellular membranes

SupramoleculeName: Ciliary rootlet surrounded by cellular membranes / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: The sample was purified according to https://doi.org/10.1016/j.cell.2012.10.038, maintaining cellular interactions with the rootlet.
Source (natural)Organism: Mus musculus (house mouse) / Organ: eye / Tissue: retina / Organelle: rootlet / Location in cell: centriole associated, cytoplasm

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.68 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFor tomogram segmentation, tomograms were reconstructed as even/odd frame half tomograms using the above mentioned WARP pipeline and denoised using Noise2Map(Tegunov & Cramer, 2018). The tomograms were then deconvolved and isotropically reconstructed with denoising using IsoNet(Liu et al, 2021) with a cube size of 128 pixels. The tomograms were then preprocessed in EMAN2.2 for training of the TomoSeg CNN(Chen et al, 2017).
Final reconstructionNumber images used: 41

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