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- EMDB-15257: Composite map of the C. reinhardtii ciliary transition zone (full... -

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Entry
Database: EMDB / ID: EMD-15257
TitleComposite map of the C. reinhardtii ciliary transition zone (full 9-fold assembly) from in situ subtomogram averaging
Map dataFull 9-fold symmetrical composite model of TZ structures including schematic representations of connecting MTDs, MTTs and central pair singlet MTs
Sample
  • Complex: Composite map of the C. reinhardtii ciliary transition zone (full 9-fold assembly)
Keywordscilia / transition zone / flagella / ciliary pore / intraflagellar transport / motility / signaling / basal body / microtubules / chlamydomonas / cytoskeleton / MTD / doublet / stellate / Y-link / sleeve / STRUCTURAL PROTEIN
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 34.7 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_15257.png

Downloads & links

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Map

FileDownload / File: emd_15257.map.gz / Format: CCP4 / Size: 1021.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull 9-fold symmetrical composite model of TZ structures including schematic representations of connecting MTDs, MTTs and central pair singlet MTs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.84 Å/pix.
x 900 pix.
= 6156. Å		6.84 Å/pix.
x 543 pix.
= 3714.12 Å		6.84 Å/pix.
x 548 pix.
= 3748.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum0.0 - 1.0000002
Average (Standard dev.)0.018617405 (±0.12129367)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions543548900
Spacing548543900
CellA: 3748.32 Å / B: 3714.12 Å / C: 6156.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 9-fold composite model: MTD Sleeve component

Fileemd_15257_additional_1.map
Annotation9-fold composite model: MTD Sleeve component
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 9-fold composite model: Stellate component (filaments only)

Fileemd_15257_additional_2.map
Annotation9-fold composite model: Stellate component (filaments only)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 9-fold composite model: Stellate component (MTDs only)

Fileemd_15257_additional_3.map
Annotation9-fold composite model: Stellate component (MTDs only)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 9-fold composite model: Y-link component

Fileemd_15257_additional_4.map
Annotation9-fold composite model: Y-link component
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Composite map of the C. reinhardtii ciliary transition zone (full...

EntireName: Composite map of the C. reinhardtii ciliary transition zone (full 9-fold assembly)
Components
  • Complex: Composite map of the C. reinhardtii ciliary transition zone (full 9-fold assembly)

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Supramolecule #1: Composite map of the C. reinhardtii ciliary transition zone (full...

SupramoleculeName: Composite map of the C. reinhardtii ciliary transition zone (full 9-fold assembly)
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: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 34.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: STOPGAP (ver. 0.3)
Details: Composite map generated from three separate subtomogram averages MTD Sleeve: 110 particles, 34.7 A Stellate: 206 particles, 28.2 A Y-link: 529 particles, 27.9 A
Number subtomograms used: 110
ExtractionNumber tomograms: 19 / Number images used: 325 / Software - Name: IMOD
Final angle assignmentType: PROJECTION MATCHING / Software - Name: STOPGAP (ver. 0.3)

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