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

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

doi:10.1016/j.cell.2023.09.017

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

Cell. 2023 Nov 9;186(23):5041-5053.e19. doi: 10.1016/j.cell.2023.09.017. Epub 2023 Oct 20.

Authors

Affiliations

¹ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA. Electronic address: zhen.chen@ucsf.edu.
² Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
³ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; J. David Gladstone Institutes, San Francisco, CA, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
⁵ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA.
⁶ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. Electronic address: valer@janelia.hhmi.org.
⁷ Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA; Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA, USA. Electronic address: david@agard.ucsf.edu.

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.

Keywords: AlphaFold2 modeling; axoneme; cellular cryo-ET; doublet; microtubule; microtubule inner protein; sperm; visual proteomics.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Axoneme / chemistry
Cryoelectron Microscopy / methods
Flagella / metabolism
Male
Mice
Microtubules / metabolism
Proteome* / analysis
Semen
Spermatozoa* / chemistry

Substances

Proteome

Abstract

Publication types

MeSH terms

Substances

Grants and funding