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- EMDB-52359: Dynamic microtubule plus end architecture in presence of EB3. -

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Basic information

Entry
Database: EMDB / ID: EMD-52359
TitleDynamic microtubule plus end architecture in presence of EB3.
Map dataExtracted subtomogram of microtubule control (free) plus end (bin 2, cryoCARE denoised).
Sample
  • Complex: Microtubules with sub-stoichiometric EB3
    • Organelle or cellular component: EB3
    • Organelle or cellular component: tubulin
Keywordsmicrotubule / plus end / protofilaments / CYTOSOLIC PROTEIN
Biological speciesSus scrofa domesticus (domestic pig) / Homo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsHoogebeen RA / Saunders HAJ / Howes SC / Akhmanova A
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO) Netherlands
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth.
Authors: Harriet A J Saunders / Cyntha M van den Berg / Robin A Hoogebeen / Donna Schweizer / Kelly E Stecker / Ronald Roepman / Stuart C Howes / Anna Akhmanova /
Abstract: Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms ...Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.
History
DepositionDec 23, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateJul 2, 2025-
Current statusJul 2, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52359.png

Downloads & links

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Map

FileDownload / File: emd_52359.map.gz / Format: CCP4 / Size: 206 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExtracted subtomogram of microtubule control (free) plus end (bin 2, cryoCARE denoised).
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AxesZ (Sec.)Y (Row.)X (Col.)
4.34 Å/pix.
x 300 pix.
= 1302. Å		4.34 Å/pix.
x 600 pix.
= 2604. Å		4.34 Å/pix.
x 300 pix.
= 1302. Å

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generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 4.34 Å
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Minimum - Maximum-0.7034308 - 0.48484677
Average (Standard dev.)-0.0036923431 (±0.047559988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600300300
Spacing300600300
CellA: 1302.0 Å / B: 2604.0 Å / C: 1302.0 Å
α=β=γ: 90.0 °

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Additional map: #1

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Additional map: Full tomogram from which main map was extracted...

Fileemd_52359_additional_26.map
AnnotationFull tomogram from which main map was extracted as a subtomogram (bin2, cryoCARE denoised).
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Sample components

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Entire : Microtubules with sub-stoichiometric EB3

EntireName: Microtubules with sub-stoichiometric EB3
Components
  • Complex: Microtubules with sub-stoichiometric EB3
    • Organelle or cellular component: EB3
    • Organelle or cellular component: tubulin

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Supramolecule #1: Microtubules with sub-stoichiometric EB3

SupramoleculeName: Microtubules with sub-stoichiometric EB3 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / Organ: brain

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Supramolecule #2: EB3

SupramoleculeName: EB3 / type: organelle_or_cellular_component / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: tubulin

SupramoleculeName: tubulin / type: organelle_or_cellular_component / ID: 3 / Parent: 1
Source (natural)Organism: Sus scrofa domesticus (domestic pig) / Organ: brain

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: MRB80 buffer = 80 mM piperazine-N, N[prime]-bis (2-ethane sulfonic acid), pH 6.8, supplemented with 4 mM MgCl2, and 1 mM EGTA.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 303.15 K / Instrument: FEI VITROBOT MARK IV
Details: Prior to vitrification, the sample was manually back-blotted inside the Vitrobot chamber..
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Cell Microscopy Core, Utrecht University Medical Center (UMC)
Diameter: 5 nm

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Average electron dose: 1.6 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 63000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Number images used: 61
CTF correctionType: NONE

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