Structural diversity of axonemes across mammalian motile cilia

Nature, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Abstract Reproduction, development and homeostasis depend on motile cilia, whose rhythmic beating is powered by a microtubule-based molecular machine called the axoneme. Although an atomic model of axoneme available for alga Chlamydomonas reinhardtii 1 , structures mammalian axonemes are incomplete 1–5 . Furthermore, we do not fully understand how vary across motile-ciliated cell types in body. Here use cryoelectron microscopy, tomography proteomics to resolve 96-nm modular repeat axonemal doublet microtubules (DMTs) from both sperm flagella epithelial cilia oviduct, brain ventricles respiratory tract. We find that DMTs most specialized, with having only minor differences tissues. build DMT, defining positions interactions 181 proteins including 34 newly identified proteins. elucidate composition radial spoke 3 uncover binding sites kinases associated regeneration ATP regulation ciliary motility. discover sperm-specific, axoneme-tethered T-complex protein ring complex (TRiC) chaperone may contribute construction or maintenance long sperm. dyneins their prestroke states, illuminating conformational changes occur during movement. Our results illustrate elements chemical mechanical embedded within axoneme, providing valuable resources understanding aetiology ciliopathy infertility, exemplifying discovery power modern structural biology.

Language: Английский

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DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models

Structure, Journal Year: 2024, Volume and Issue: 32(8), P. 1248 - 1259.e5

Published: May 15, 2024

Language: Английский

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Advances in cryo-ET data processing: meeting the demands of visual proteomics

Current Opinion in Structural Biology, Journal Year: 2024, Volume and Issue: 87, P. 102861 - 102861

Published: June 17, 2024

Cryogenic electron tomography (cryo-ET), a method that enables the viewing of biomolecules in near-native environments at high resolution, is rising accessibility and applicability. Over past several years, once slow sample preparation data collection procedures have seen innovations which enable rapid large datasets required for attaining resolution structures. Increased availability has provided driving force exciting improvements cryo-ET processing methodologies throughout entire pipeline development accessible graphical user interfaces (GUIs) individuals inexperienced computational fields to convert raw tilt series into 3D These advances are enabling attain higher extending its applicability more complex samples.

Language: Английский

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Expanding insights from in situ cryo-EM

Current Opinion in Structural Biology, Journal Year: 2024, Volume and Issue: 88, P. 102885 - 102885

Published: July 11, 2024

Language: Английский

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DiffModeler: large macromolecular structure modeling for cryo-EM maps using a diffusion model

Nature Methods, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 21, 2024

Language: Английский

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DiffModeler: Large Macromolecular Structure Modeling in Low-Resolution Cryo-EM Maps Using Diffusion Model

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 23, 2024

Cryogenic electron microscopy (cryo-EM) has now been widely used for determining multi-chain protein complexes. However, modeling a complex structure is challenging particularly when the map resolution low, typically in intermediate range of 5 to 10 Å. Within this range, even accurate fitting difficult, let alone de novo modeling. To address challenge, here we present DiffModeler, fully automated method structures. DiffModeler employs diffusion model backbone tracing and integrates AlphaFold2-predicted single-chain structures fitting. Extensive testing on cryo-EM maps at resolutions demonstrates exceptional accuracy modeling, achieving an average TM-Score 0.92, surpassing existing methodologies significantly. Notably, successfully modeled composed 47 chains 13,462 residues, high 0.94. Further benchmarking low (10-20 Å confirms its versatility, demonstrating plausible performance. Moreover, coupled with CryoREAD, excels constructing protein-DNA/RNA near-atomic (0-5 Å), showcasing state-of-the-art performance TM-Scores 0.88 0.91 across two datasets.

Language: Английский

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Tektin bundle interacting protein, TEKTIP1, functions to stabilize the tektin bundle and axoneme in mouse sperm flagella

Cellular and Molecular Life Sciences, Journal Year: 2024, Volume and Issue: 81(1)

Published: March 7, 2024

Abstract Tektins are microtubule inner proteins (MIPs) and localize at the inside lumen of doublet microtubules (DMTs) cilia/flagella. TEKTIP1, a newly identified protein by cryo-electron microscopy (cryo-EM), is proposed to be localized center tektin bundle hypothesized recruit tektins or stabilize bundle. However, physiological role TEKTIP1 unknown. In this study, we generated Tektip1 -knockout ( −/− ) mice showed that they were male subfertile primarily due reduced sperm motility. A high percentage from moderately disorganized axoneme structures abnormal flagellar waveforms. predominately interacted with TEKT3 among tektins. Loss partially disturbed organization mainly affecting native status its interaction other Collectively, our study reveals potential molecular mechanism in axonemal structure motility, highlights importance MIPs stabilizing DMTs, suggests relevance deficiency human asthenospermia. will an excellent animal model DMT flagella using cryo-EM future.

Language: Английский

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Disruption of the mitochondrial network in a mouse model of Huntington's disease visualized by in-tissue multiscale 3D electron microscopy

Acta Neuropathologica Communications, Journal Year: 2024, Volume and Issue: 12(1)

Published: June 5, 2024

Abstract Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by expanded CAG repeat in the coding sequence of huntingtin protein. Initially, it predominantly affects medium-sized spiny neurons (MSSNs) corpus striatum. No effective treatment still available, thus urging identification potential therapeutic targets. While evidence mitochondrial structural alterations HD exists, previous studies mainly employed 2D approaches and were performed outside strictly native brain context. In this study, we adopted a novel multiscale approach to conduct comprehensive 3D situ analysis disturbances mouse model HD. We investigated MSSNs within tissue under optimal conditions utilizing state-of-the-art imaging technologies, specifically FIB/SEM for complete neuronal somas Electron Tomography detailed morphological examination, image processing-based quantitative analysis. Our findings suggest disruption network towards fragmentation The interlaced, slim long mitochondria observed healthy transforms into isolated, swollen short entities, with internal cristae disorganization, cavities abnormally large matrix granules.

Language: Английский

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Structural insights into the diversity and DNA cleavage mechanism of Fanzor

Cell, Journal Year: 2024, Volume and Issue: 187(19), P. 5238 - 5252.e20

Published: Aug. 28, 2024

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe structures of Fzs from three different organisms. We find that share a common ωRNA interaction interface, regardless length ωRNA, which varies considerably across species. The analysis also reveals Fz's mode DNA recognition and unwinding capabilities as well presence non-canonical catalytic site. demonstrate how protein conformations Fz shift to allow binding double-stranded active site within R-loop. Mechanistically, examination in states shows conformation lid loop on RuvC controlled by formation guide/DNA heteroduplex, regulating activation nuclease displacement at single cleavage Our findings clarify mechanism Fz, establishing foundation for engineering efforts.

Language: Английский

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Frontiers in integrative structural modeling of macromolecular assemblies

QRB Discovery, Journal Year: 2025, Volume and Issue: 6

Published: Jan. 1, 2025

Abstract Integrative modeling enables structure determination for large macromolecular assemblies by combining data from multiple experiments with theoretical and computational predictions. Recent advancements in AI-based prediction cryo electron-microscopy have sparked renewed enthusiasm integrative modeling; structures methods can be integrated situ maps to characterize assemblies. This approach previously allowed us others determine the architectures of diverse assemblies, such as nuclear pore complexes, chromatin remodelers, cell–cell junctions. Experimental spanning several scales was used these studies, ranging high-resolution data, X-ray crystallography AlphaFold structure, low-resolution cryo-electron tomography co-immunoprecipitation experiments. Two recurrent challenges emerged across a range studies. First, contained significant fractions disordered regions, necessitating development new regions context ordered regions. Second, needed developed utilize information tomography, timely challenge structural biology is increasingly moving towards characterization. Here, we recapitulate recent developments proteins analysis highlight other opportunities method modeling.

Language: Английский

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