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- EMDB-49936: CLEM Cilium N17 for IFT motion study -

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Basic information

Entry
Database: EMDB / ID: EMD-49936
TitleCLEM Cilium N17 for IFT motion study
Map data
Sample
  • Organelle or cellular component: Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88
KeywordsIFT / microtubule / mammalian primary cilia / 3D structure / TRANSPORT PROTEIN
Biological speciesMus musculus (house mouse)
Methodelectron tomography / negative staining
AuthorsSun S / Liang B / Koplas A / Tikhonenko I / Nachury M / Khodjakov A / Sui H
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM143223 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM152716 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130298 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Intraflagellar transport trains can switch rails and move along multiple microtubules in intact primary cilia.
Authors: Shufeng Sun / Biqing Liang / Adam Koplas / Irina Tikhonenko / Maxence Nachury / Alexey Khodjakov / Haixin Sui /
Abstract: Structural homeostasis and proper distributions of signaling molecules in cilia require a constant flow of cargoes carried by intraflagellar transport (IFT) trains in both anterograde and retrograde ...Structural homeostasis and proper distributions of signaling molecules in cilia require a constant flow of cargoes carried by intraflagellar transport (IFT) trains in both anterograde and retrograde directions within the thin, long ciliary shafts. In the motile cilium framework, the nine microtubule doublets of the same length serve as the transportation rails, and a preferential association to the two subtubules of the microtubule doublets prevents collisions among the IFT trains that move in opposite directions. However, this mechanism is incompatible with the primary cilia structure, where most of the nine microtubule doublets terminate in the ciliary shafts-only several of them reach the ciliary tip and only in a singlet form. Here, we demonstrate that anterograde and retrograde trains in primary cilia interact with both subtubules of the microtubule doublets without apparent preference. They can switch microtubules, and they may simultaneously interact with multiple microtubules to facilitate their movement. This architecture makes the collisions inevitable, and live-cell recordings reveal that anterograde and retrograde trains tend to pause when they come into direct contact. We also find that the velocity of the train's movement often changes after the pause. Thus, the motion behaviors of IFT trains in primary cilia are distinctive from those of motile cilia, and our data offer an essential foundation for understanding proper signaling molecule distributions in primary cilia.
History
DepositionMar 28, 2025-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 30, 2025-
Current statusApr 30, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49936.png

Downloads & links

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Map

FileDownload / File: emd_49936.map.gz / Format: CCP4 / Size: 17.8 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10 Å/pix.
x 518 pix.
= 5180. Å		10 Å/pix.
x 996 pix.
= 9960. Å		10 Å/pix.
x 708 pix.
= 7080. Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10.624 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-32768.0 - 32767.0
Average (Standard dev.)5255.194999999999709 (±3717.497800000000097)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions76866561870
Spacing66567681870
CellA: 70713.34 Å / B: 8159.2314 Å / C: 19866.879 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88

EntireName: Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88
Components
  • Organelle or cellular component: Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88

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Supramolecule #1: Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88

SupramoleculeName: Primary cilium of mIMCD3 cells with triple-mNeonGreen-fusion-IFT88
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: This cilium correlates to timed IFT motion tracked by triple-mNeonGreen-fusion-IFT88. This study resulted in kymographic (LM) analysis and subsequent ultrastructure shown here.
Source (natural)Organism: Mus musculus (house mouse) / Organ: Kidney / Organelle: Primary Cilium

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4 / Component - Concentration: 1.0 X / Component - Formula: PBS / Component - Name: Phosphate-buffered saline
Details: NaCl: 137 mM KCl: 2.7 mM Na2HPO4: 10 mM KH2PO4: 1.8 mM
StainingType: POSITIVE / Material: Uranyl Acetate, Lead Citrate
Details: The sections were stained with Uranyl acetate followed by lead citrate solution.
Sugar embeddingMaterial: SPI-Pon812
Details: Samples were dehydrated post fixation using an ethanol gradient at RT. Specimens were then back-substituted with acetone. After infiltration with resin the samples were cured.
GridModel: Homemade / Material: COPPER / Support film - Material: FORMVAR / Support film - topology: CONTINUOUS
DetailsSpecimen is mIMCD3 cells labeled with triple-mNeonGreen-fusion-IFT88.
Cryo protectantno
SectioningUltramicrotomy - Instrument: UC6 / Ultramicrotomy - Temperature: 293 K / Ultramicrotomy - Final thickness: 120
Ultramicrotomy - Details: The samples in the resin block were sectioned into serial sections with a target thickness of 150 nm. Most of the sections had an actual thickness of around 120 nm.
Fiducial markerManufacturer: Sigma / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 292.0 K / Max: 298.0 K
DetailsA Philips High tilt specimen holder was used for data collection purposes.
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 6940 / Average electron dose: 15.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 4.2 µm / Calibrated defocus min: 3.5 µm / Calibrated magnification: 19000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 19000
Sample stageSpecimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D / Number images used: 6940

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