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- EMDB-15258: In situ subtomogram average of C. reinhardtii IFT-B at the ciliar... -

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Basic information

Entry
Database: EMDB / ID: EMD-15258
TitleIn situ subtomogram average of C. reinhardtii IFT-B at the ciliary transition zone
Map data
Sample
  • Complex: IFT-B at the C. reinhardtii ciliary transition zone
Keywordscilia / transition zone / flagella / ciliary pore / intraflagellar transport / motility / signaling / basal body / microtubules / chlamydomonas / membrane proteins / membrane / trafficking / IFT trains / IFT / kinesin / dynein / motor protein / STRUCTURAL PROTEIN
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 24.4 Å
Authorsvan den Hoek HG / Jordan MA / Alvarez Viar G / Righetto RD / Schaffer M / Erdmann PS / Wan WN / Plitzko JM / Baumeister W / Pigino G / 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_15258.png

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Map

FileDownload / File: emd_15258.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
6.84 Å/pix.
x 128 pix.
= 875.52 Å		6.84 Å/pix.
x 128 pix.
= 875.52 Å		6.84 Å/pix.
x 128 pix.
= 875.52 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 6.84 Å
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Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.14781784 - 0.22935544
Average (Standard dev.)-0.000048337766 (±0.014474459)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 875.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15258_msk_1.map
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Half map: #1

Fileemd_15258_half_map_1.map
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Half map: #2

Fileemd_15258_half_map_2.map
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Sample components

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Entire : IFT-B at the C. reinhardtii ciliary transition zone

EntireName: IFT-B at the C. reinhardtii ciliary transition zone
Components
  • Complex: IFT-B at the C. reinhardtii ciliary transition zone

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Supramolecule #1: IFT-B at the C. reinhardtii ciliary transition zone

SupramoleculeName: IFT-B at the C. reinhardtii 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 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 100.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination 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: 42000
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 reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: STOPGAP (ver. 0.3) / Number subtomograms used: 1361
ExtractionNumber tomograms: 19 / Number images used: 5715
Final angle assignmentType: PROJECTION MATCHING / Software - Name: STOPGAP (ver. 0.3)

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