De Novo Protein Identification in Mammalian Sperm Using in Situ Cryoelectron Tomography and AlphaFold2 Docking

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2023 Oct 21

PMID 37865089

Authors

Zhen Chen

Momoko Shiozaki

Kelsey M Haas

Will M Skinner

Shumei Zhao

Caiying Guo

Benjamin J Polacco

Zhiheng Yu

Nevan J Krogan

Polina V Lishko

Robyn M Kaake

Ronald D Vale

David A Agard

Affiliations

Soon will be listed here.

Abstract

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-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.

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