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- EMDB-48296: Structure of axonemal DMT from Tetrahymena thermophila RIB72A/Del... -

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Entry
Database: EMDB / ID: EMD-48296
TitleStructure of axonemal DMT from Tetrahymena thermophila RIB72A/Delta_EF-hand mutant, centered on the A-tubule RIB72A/B in 48-nm repeat
Map dataStructure of axonemal DMT from Tetrahymena thermophila RIB72A/Delta_EF-hand mutant, centered on the A-tubule RIB72A/B in 48-nm repeat
Sample
  • Organelle or cellular component: Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta_EF-hand truncation
Keywordscilia / flagellum / STRUCTURAL PROTEIN
Biological speciesTetrahymena thermophila (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 7.57 Å
AuthorsLi S / Winey M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127571 United States
CitationJournal: Mol Biol Cell / Year: 2025
Title: A ternary complex of MIPs in the A-tubule of basal bodies and axonemes depends on RIB22 and the EF-hand domain of RIB72A in cilia.
Authors: Rachel A Howard-Till / Sam Li / Usha Pallabi Kar / Christopher N Fuentes / Amy S Fabritius / Mark Winey /
Abstract: The lumens of the highly stable microtubules that make up the core of basal bodies, cilia, and flagella are coated with a network of proteins known as MIPs, or microtubule inner proteins. MIPs are ...The lumens of the highly stable microtubules that make up the core of basal bodies, cilia, and flagella are coated with a network of proteins known as MIPs, or microtubule inner proteins. MIPs are hypothesized to enhance the rigidity and stability of these microtubules, but how they assemble and contribute to cilia function is poorly understood. Here we describe a ciliate specific MIP, RIB22, in . RIB22 is a calmodulin-like protein found in the A-tubule of doublet and triplet microtubules in cilia and basal bodies. Its localization is dependent on the conserved MIP RIB72. Here we use cryogenic electron tomography (cryoET) to examine RIB22 and its interacting partners in axonemes and basal bodies. RIB22 forms a ternary complex with the C-terminal EF-hand domain of RIB72A and another MIP, FAM166A. strains lacking RIB22 or the EF-hand domain of RIB72A showed impaired cilia function. CryoET on axonemes from these strains demonstrated an interdependence of the three proteins for stabilization within the structure. Deletion of the RIB72A EF-hand domain resulted in the apparent loss of multiple MIPs in the region. These findings emphasize the intricacy of the MIP network and the importance of understanding MIPs' functions during cilium assembly and regulation.
History
DepositionDec 13, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48296.png

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Map

FileDownload / File: emd_48296.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of axonemal DMT from Tetrahymena thermophila RIB72A/Delta_EF-hand mutant, centered on the A-tubule RIB72A/B in 48-nm repeat
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
2.65 Å/pix.
x 220 pix.
= 583. Å		2.65 Å/pix.
x 220 pix.
= 583. Å		2.65 Å/pix.
x 220 pix.
= 583. Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.65 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.0528
Minimum - Maximum-0.12251958 - 0.2109525
Average (Standard dev.)0.00077285967 (±0.01091959)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-220
Dimensions220220220
Spacing220220220
CellA=B=C: 583.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48296_msk_1.map
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Half map: Half map 2

Fileemd_48296_half_map_1.map
AnnotationHalf map 2
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Half map: Half map 1

Fileemd_48296_half_map_2.map
AnnotationHalf map 1
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Sample components

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Entire : Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta...

EntireName: Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta_EF-hand truncation
Components
  • Organelle or cellular component: Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta_EF-hand truncation

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Supramolecule #1: Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta...

SupramoleculeName: Axoneme isolated from Tetrahymena thermophila mutant RIB72A/Delta_EF-hand truncation
type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Tetrahymena thermophila (eukaryote)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 1xTE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 78.0 K / Max: 78.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number real images: 1 / Average exposure time: 0.36 sec. / Average electron dose: 1.33 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 18868 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 33000
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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number subtomograms used: 8737
ExtractionNumber tomograms: 29 / Number images used: 15541
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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