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

Understanding molecular mechanisms of cellular pathways 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 and subtomogram averaging provided 6.0-angstrom resolution reconstructions of axonemal microtubules and their associated proteins. The well-resolved secondary and tertiary structures allowed us to computationally match, in an unbiased manner, novel densities in our 3D reconstruction maps with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105 and SPACA9 as novel microtubule inner proteins that form an extensive network crosslinking the lumen of microtubule and existing proteins. Additional biochemical and mass spectrometry analyses helped validate potential candidates. The novel axonemal sperm structures identified by this approach form an extensive interaction network within the lumen of microtubules, suggesting they have a role in the mechanical and elastic properties of the microtubule filaments required for the vigorous beating motions of flagella. ### Competing Interest Statement We declare that one or more authors have a competing interest as defined by Nature Portfolio. The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, GEn1E Lifesciences, Inc. and Twist Bioscience Corp. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics. The authors have no conflicts of interest to declare that are relevant to the content of this article.