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- EMDB-15253: In situ subtomogram average of the C. reinhardtii Y-link at the c... -

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Basic information

Entry
Database: EMDB / ID: EMD-15253
TitleIn situ subtomogram average of the C. reinhardtii Y-link at the ciliary transition zone
Map data
Sample
  • Complex: Y-link at the ciliary transition zone
Keywordscilia / transition zone / flagella / ciliary pore / intraflagellar transport / motility / signaling / basal body / microtubules / chlamydomonas / STRUCTURAL PROTEIN
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 27.9 Å
Authorsvan den Hoek HG / Righetto RD / Schaffer M / Erdmann PS / Wan WN / Plitzko JM / Baumeister W / Engel BD
Funding supportEuropean Union, Germany, Switzerland, 7 items
OrganizationGrant numberCountry
European Molecular Biology Organization (EMBO)ALTF 537-2021European Union
Alexander von Humboldt Foundation Germany
European Research Council (ERC)819826European Union
European Research Council (ERC)715289European Union
Swiss National Science FoundationPP00P3_187198 Switzerland
Max Planck Society Germany
Helmholtz Association Germany
CitationJournal: Science / Year: 2022
Title: In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains.
Authors: Hugo van den Hoek / Nikolai Klena / Mareike A Jordan / Gonzalo Alvarez Viar / Ricardo D Righetto / Miroslava Schaffer / Philipp S Erdmann / William Wan / Stefan Geimer / Jürgen M Plitzko / ...Authors: Hugo van den Hoek / Nikolai Klena / Mareike A Jordan / Gonzalo Alvarez Viar / Ricardo D Righetto / Miroslava Schaffer / Philipp S Erdmann / William Wan / Stefan Geimer / Jürgen M Plitzko / Wolfgang Baumeister / Gaia Pigino / Virginie Hamel / Paul Guichard / Benjamin D Engel /
Abstract: The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the ...The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in . We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.
History
DepositionJun 24, 2022-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15253.png

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Map

FileDownload / File: emd_15253.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 6.84 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.16565357 - 0.20526677
Average (Standard dev.)0.00009982894 (±0.011593105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 1313.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15253_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Half map: #1

Fileemd_15253_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15253_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Y-link at the ciliary transition zone

EntireName: Y-link at the ciliary transition zone
Components
  • Complex: Y-link at the ciliary transition zone

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Supramolecule #1: Y-link at the ciliary transition zone

SupramoleculeName: Y-link at the ciliary transition zone / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC-3994 / Organelle: Cilium

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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: 7
GridModel: Quantifoil R2/1 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 100.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 19 / Number images used: 1873
Final angle assignmentType: PROJECTION MATCHING / Software - Name: STOPGAP (ver. 0.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 27.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: STOPGAP (ver. 0.3) / Number subtomograms used: 529

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