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- EMDB-15978: Masked refinement of the IFTB1 subcomplex within anterograde intr... -

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Entry
Database: EMDB / ID: EMD-15978
TitleMasked refinement of the IFTB1 subcomplex within anterograde intraflagellar trains in Chlamydomonas reinhardtii cilia
Map data
Sample
  • Complex: Two IFTB repeats from anterograde intraflagellar transport trains within native Chlamydomonas reinhardtii cilia
KeywordsCilia / IFT / Intraflagellar / transport / PROTEIN TRANSPORT
Biological speciesChlamydomonas reinhardtii (plant)
Methodsubtomogram averaging / cryo EM / Resolution: 9.9 Å
AuthorsLacey SE / Pigino G
Funding supportEuropean Union, Italy, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
Other government Italy
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: The molecular structure of IFT-A and IFT-B in anterograde intraflagellar transport trains.
Authors: Samuel E Lacey / Helen E Foster / Gaia Pigino /
Abstract: Anterograde intraflagellar transport (IFT) trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFT-A and IFT-B proteins that link structural and signaling cargos to ...Anterograde intraflagellar transport (IFT) trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFT-A and IFT-B proteins that link structural and signaling cargos to microtubule motors for import into cilia. It remains unknown how the IFT-A/-B proteins are arranged into complexes and how these complexes polymerize into functional trains. Here we use in situ cryo-electron tomography of Chlamydomonas reinhardtii cilia and AlphaFold2 protein structure predictions to generate a molecular model of the entire anterograde train. We show how the conformations of both IFT-A and IFT-B are dependent on lateral interactions with neighboring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. Following three-dimensional classification, we reveal how IFT-B extends two flexible tethers to maintain a connection with IFT-A that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that govern cilia assembly.
History
DepositionOct 18, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15978.png

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Map

FileDownload / File: emd_15978.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 3.03 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.7016319 - 1.5081921
Average (Standard dev.)0.0012685305 (±0.036089282)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 775.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15978_msk_1.map
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AxesZYX

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Additional map: A masked refinement on the IFTB1 periphery region,...

Fileemd_15978_additional_1.map
AnnotationA masked refinement on the IFTB1 periphery region, containing the regions corresponding to IFT56 and IFT52/46 heterodimer. Lowpassed to 16 angstrom and with a -1600 A^2 bfactor
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Half map: #1

Fileemd_15978_half_map_1.map
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Half map: #2

Fileemd_15978_half_map_2.map
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Sample components

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Entire : Two IFTB repeats from anterograde intraflagellar transport trains...

EntireName: Two IFTB repeats from anterograde intraflagellar transport trains within native Chlamydomonas reinhardtii cilia
Components
  • Complex: Two IFTB repeats from anterograde intraflagellar transport trains within native Chlamydomonas reinhardtii cilia

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Supramolecule #1: Two IFTB repeats from anterograde intraflagellar transport trains...

SupramoleculeName: Two IFTB repeats from anterograde intraflagellar transport trains within native Chlamydomonas reinhardtii cilia
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#13
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statehelical array

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Sample preparation

BufferpH: 7 / Details: TAP (Tris-Acetate-Phosphate) Media
VitrificationCryogen name: ETHANE
DetailsChlamydomonas reinhardtii cells applied to quantifoil grids and plunge frozen; cilia project out from cell bodies and traverse holes in the carbon film.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 2.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: RELION (ver. 3.1.3), Warp) / Number subtomograms used: 18216
ExtractionNumber tomograms: 600 / Number images used: 18216
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT

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