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Concise assessment

This Nature paper presents near-complete 96-nm atomic models of mammalian axonemal DMTs (including bovine sperm, bovine oviduct, porcine brain ventricles and human oviduct), identifies ~34 new sperm DMT proteins, reveals a sperm-specific RS-tethered TRiC chaperone and RS3 enzyme/kinase architecture, and resolves dyneins in prestroke conformations — providing a high-quality structural atlas directly relevant to ciliopathy and infertility genetics

Key claims in the manuscript are directly supported by large cryo-EM/spa and cryo-ET datasets, proteomics (PRIDE PXD035941) and deposited EMDB/PDB maps/models for independent validation

Representative citation

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Detailed paper review and critique

1) What the paper did and why it matters

The authors combined large-scale single particle analysis cryo-EM, cryo-ET subtomogram averaging and complementary proteomics to reconstruct composite 96-nm repeats of doublet microtubules (DMTs) from multiple mammalian motile cilia types (bovine sperm, bovine oviduct, porcine brain ventricle and human oviduct). They produced an atomic model of the bovine sperm 96-nm repeat (PDB 9FQR) and deposited EM maps (EMDB entries including EMD-50664 etc.), used AlphaFold2 and structure-based searches to assign previously unidentified densities, and integrated MS proteomics (PRIDE PXD035941) to validate protein identities and tissue specificity. Their core conclusion is that sperm DMTs have additional layers of complexity (~30 sperm-specific proteins) including a TRiC chaperone tethered to radial spokes, extra MIPs and kinases that create local ATP regeneration hubs, and that dynein motors adopt prestroke conformations captured in the maps — with direct implications for the molecular bases of male infertility and ciliopathies

Primary supporting text from the paper

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2) Strengths

• Scale and data depth: very large SPA dataset (45,431 micrographs/videos for bovine sperm combined with prior data) enabling high-resolution reconstructions and local focused refinements across 39 subregions as described in Methods Dataset
• Multimodal validation: cryo-ET subtomogram averages from porcine oviducts corroborate SPA reconstructions and the authors deposit corresponding EMDB accessions for independent checks SPA
• Data transparency: EMDB and PDB deposits (for example PDB 9FQR and EMDB EMD-50664) and PRIDE proteomics make independent reanalysis straightforward Data
• Methodological rigor: sophisticated focused classification, tubulin signal subtraction, AlphaFold2-guided assignments and reverse searches to raise confidence in density assignments reduce misassignment risk Protein

3) Limitations, blindspots and potential biases

• Consensus averaging and splaying: The authors state that consensus maps represent averages of all nine DMTs and splaying causes loss of positional information; this can obscure asymmetry and low-occupancy features, potentially undercounting heterogeneity (paper notes several asymmetric complexes remain unresolved) Consensus
• Assignment confidence: many newly assigned proteins rely on fitted AlphaFold models and cross-correlation ranking; while structure-guided assignment is state-of-the-art, without orthogonal biochemical validation (e.g., antibody localization, targeted MS of isolated complexes or genetics) some assignments remain putative — authors acknowledge alternatives in Supplementary Figs 8-33 AlphaFold
• Functional inference gap: structural observation of TRiC tethering, AKs, and kinases is compelling, but the paper notes experimental tests (e.g., in vivo TRiC inhibitors) are unavailable and functional causality remains to be proven Functional
• Species and tissue sampling: coverage is excellent across several mammals and tissues but evolutionary claims (e.g., when TRiC became associated with sperm axoneme or links to fertilization mode) require broader taxon sampling, particularly non-model and external fertilizing species (authors note need for structural studies of non-model organisms) Evolutionary

4) Reproducibility and resources for follow-up

The authors provided code and data availability links including GitHub repository Doublet and multiple EMDB/PDB deposits plus a PRIDE proteomics accession (PXD035941). Methods are detailed (cryoSPARC, RELION, FREALIGN, focused classification strategy, proteomics pipelines). This high degree of data sharing is excellent for reproducibility; however successful reproduction will need substantial EM/ET expertise and computational resources (they used custom scripts for alignment correction and multi-box stitching due to box-size tradeoffs) Data

5) Biological and clinical implications

• Sperm-specific proteins identified are plausible candidate loci for male-specific ciliopathies and isolated infertility; authors highlight this and suggest candidates for contraceptive targets — but functional genetics will be required to link variants to human phenotypes Fertility
• Local ATP regeneration hubs anchored to RSs (adenylate kinases AK7/AK8/AK9, PKA holoenzyme, CAMK4) suggest biochemical compartmentalization within radial spokes that could tune beat mechanics — a testable mechanistic hypothesis using activity mutants or localized inhibitors ATP

6) Specific technical and conceptual suggestions for follow-up experiments

1. Genetic perturbation: CRISPR knockout or conditional deletion of selected sperm-specific proteins (for example ARMH1, WDR64, RS3 components, CCT6B/CCT8L2) in mouse to test assembly, flagellar waveform, and fertility — especially selecting proteins with clear unique density assignments and detectable MS peptides
2. Biochemical pulldowns: affinity purification of radial spoke complexes from sperm tails followed by targeted MS and crosslinking-MS to independently confirm proximity and stoichiometry of TRiC and AK complexes
3. Functional assays: live imaging of beat frequency/waveform in cilia engineered to express or lack specific sperm proteins (exogenous expression in epithelial cilia or knock-in of sperm paralogues) to directly link structure to function
4. Higher-resolution targeted cryo-ET: focus on TRiC–RS interface using in situ cryo-ET of intact sperm to resolve lumenal densities and tethering elements currently unresolved because of averaging

7) Confidence and where evidence could change conclusions

Confidence in the structural assignments and the overall conclusion that sperm DMTs are more specialized is high because of the dataset scale, multiple species/tissue types, and the data deposition that allow verification. However, the functional interpretation of TRiC as a maintenance/refolding chaperone or as a mechanical coupler is a mechanistic hypothesis that would be overturned if targeted loss-of-function of TRiC tethering did not alter tubulin turnover, axonemal stability or sperm motility. Similarly, if future higher-resolution maps or orthogonal biochemistry reassign some densities to different proteins, the number of sperm-specific proteins and certain interaction networks may change — the authors explicitly acknowledge such possibilities TRiC

8) Short checklist for researchers wanting to reuse these data

• Download EM maps from EMDB accessions listed in Data availability and use PDB 9FQR coordinates for direct model/map comparisons
• Obtain PRIDE dataset PXD035941 for proteomic peptide evidence when building crosslinks or selecting gene targets for genetics
• Inspect Supplementary Figs 8-33 where alternative assignments are listed before designing perturbation experiments

9) Final critical verdict

The paper is a high-quality, data-rich structural atlas of mammalian motile cilia with substantial novelty (discovery of sperm-specific TRiC tethering, RS3 composition, and axonemal enzyme localization). The main limitations are the usual ones for large cryo-EM atlases: consensus averaging and reliance on structure-prediction-guided assignments leave a residual uncertainty that will only be resolved by orthogonal biochemical and genetic experiments. Nevertheless, the resource will be highly valuable to cell biologists and clinicians studying ciliopathies and male infertility.

10) Quick actionable next steps for the community

1. Target top candidate sperm-specific proteins for knockout and phenotype assays in mouse
2. Perform affinity purification and crosslinking-MS of radial spoke complexes to validate subunit composition
3. Acquire higher-resolution focused cryo-ET of TRiC–RS interfaces

Useful resources and links

• Visualize EMDB Deposits
• Author Review Miguel Ricardo Leung

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End of review