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- EMDB-46490: The cryo-EM structure of RS3 of mouse respiratory cilia -

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Basic information

Entry
Database: EMDB / ID: EMD-46490
TitleThe cryo-EM structure of RS3 of mouse respiratory cilia
Map dataStructure of RS3 of mouse respiratory cilia
Sample
  • Organelle or cellular component: RS3 from mouse respiratory cilia
Keywordsaxoneme / radial spoke / STRUCTURAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsYanhe Z / Xuewu Z / Daniela N
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM083122 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR140082 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130289 United States
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Mouse radial spoke 3 is a metabolic and regulatory hub in cilia.
Authors: Yanhe Zhao / Kangkang Song / Amirrasoul Tavakoli / Long Gui / Angeles Fernandez-Gonzalez / Song Zhang / Petras P Dzeja / S Alex Mitsialis / Xuewu Zhang / Daniela Nicastro /
Abstract: Cilia are microtubule-based organelles that have important roles in cell sensing, signaling and motility. Recent studies have revealed the atomic structures of many multicomponent ciliary complexes, ...Cilia are microtubule-based organelles that have important roles in cell sensing, signaling and motility. Recent studies have revealed the atomic structures of many multicomponent ciliary complexes, elucidating their mechanisms of action. However, little is known about the structure, proteome and function of full-length radial spoke 3 (RS3), a conserved complex that transmits mechanochemical signals to coordinate ciliary motility. Here, we combined single-particle cryo-electron microscopy, cryo-electron tomography, proteomic analysis and computational modeling to determine the three-dimensional structure and atomic model of RS3 from mouse respiratory cilia. We reveal all RS3 components, including regulatory and metabolic enzymes such as a protein kinase A subunit, adenylate kinases (AKs) and malate dehydrogenases. Furthermore, we confirm RS3 loss in AK7-deficient mice, which exhibit motility defects. Our findings identify RS3 as an important regulatory and metabolic hub that maintains sufficient adenosine triphosphate for sustained ciliary beating, providing insights into the etiology of ciliopathies.
History
DepositionAug 8, 2024-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateFeb 25, 2026-
Current statusFeb 25, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46490.png

Downloads & links

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Map

FileDownload / File: emd_46490.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of RS3 of mouse respiratory cilia
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 360 pix.
= 776.88 Å		2.16 Å/pix.
x 360 pix.
= 776.88 Å		2.16 Å/pix.
x 360 pix.
= 776.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.158 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0016
Minimum - Maximum-0.013632098 - 0.03599633
Average (Standard dev.)0.000013370675 (±0.0002577432)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 776.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map A

Fileemd_46490_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B

Fileemd_46490_half_map_2.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RS3 from mouse respiratory cilia

EntireName: RS3 from mouse respiratory cilia
Components
  • Organelle or cellular component: RS3 from mouse respiratory cilia

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Supramolecule #1: RS3 from mouse respiratory cilia

SupramoleculeName: RS3 from mouse respiratory cilia / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#29
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 30 mM HEPES pH 7.4, 25 mM KCl, 5 mM MgSO4, 1 mM EGTA, 0.1 mM EDTA
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE
Details: 4 microliters of splayed axoneme sample were applied to glow-discharged Quantifoil R2/2 Cu grids. After removing excess liquid by blotting the grid from the backside with Whatman filter ...Details: 4 microliters of splayed axoneme sample were applied to glow-discharged Quantifoil R2/2 Cu grids. After removing excess liquid by blotting the grid from the backside with Whatman filter paper for 2-3 s, the grid was immediately plunge-frozen in liquid ethane using a home-made plunge freezer..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number real images: 50000 / Average exposure time: 4.2 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Particle selectionNumber selected: 280000
Details: particles containing RS3 were manually picked (using the conserved spacing of RSs as guide) centering on the A-tubule of the DMT
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: For the RS3 head, neck, and stalk, the AlphaFold2 models of mouse were used.
Details: For the RS3 base, the PDB entry 8J07 was used as starting.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 191448
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 3.3.1)

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