Dataset Information

De novo protein identification in mammalian sperm using high-resolution in situ cryo-electron tomography

ABSTRACT: Understanding molecular mechanisms of cellular pathways regularly requires knowledge of the identities of participating proteins, their cellular localization and their 3D structures. Contemporary workflows typically require multiple techniques to identify target proteins, track their localization using fluorescence microscopy, followed by in vitro structure determination. To identify mammal-specific sperm proteins and understand their functions, we developed a visual proteomics workflow to directly address these challenges. Our in situ cryo-electron tomography analyses of mouse and human sperm revealed key microtubule structures at 6.0 Å resolution via subtomogram averaging. The well-resolved secondary and tertiary structures allowed us to unbiasedly match novel densities discovered in our 3D reconstruction maps with 21615 protein models from the library of the mouse proteome generated by AlphaFold2. Additional biochemical and mass spectrometry analyses helped validate potential candidates. Not only was it possible to de novo identify novel mammal-specific sperm proteins using this label-free structural approach, but their cellular localizations and molecular interactions could be determined in situ. Specifically, the novel sperm proteins form an extensive interaction network crosslinking the lumen of microtubules, suggesting they could modulate the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella. This project includes global protein abundance data from five biochemical fractionations of mouse sperm cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Testis, Sperm

SUBMITTER: Robyn Kaake

LAB HEAD: Robyn Kaake

PROVIDER: PXD036885 | Pride | 2024-05-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	ProteinGroups.txt	Txt
	SearchGroup1Evidence.txt	Txt
	TableS1_LCandMSParameters.xlsx	Xlsx
	Uniprot_Musmusculus_reviewed_05072022_plusC15orf65.fasta	Fasta
	ZCGlobalAbundance_Metadata_08182022.xlsx	Xlsx

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Publications

De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.

Chen Zhen Z Shiozaki Momoko M Haas Kelsey M KM Skinner Will M WM Zhao Shumei S Guo Caiying C Polacco Benjamin J BJ Yu Zhiheng Z Krogan Nevan J NJ Lishko Polina V PV Kaake Robyn M RM Vale Ronald D RD Agard David A DA

Cell 20231020 23

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well- ...[more]

PMID: 37865089

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