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TitleDe novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.
Journal, issue, pagesCell, Vol. 186, Issue 23, Page 5041-5053.e19, Year 2023
Publish dateNov 9, 2023
AuthorsZhen 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 /
PubMed AbstractTo understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their ...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.
External linksCell / PubMed:37865089 / PubMed Central
MethodsEM (subtomogram averaging)
Resolution6.0 - 10.4 Å
Structure data

EMDB-41315: 16-nm repeating structure of A-tubule from mouse sperm flagella
Method: EM (subtomogram averaging) / Resolution: 6.0 Å

EMDB-41316: 16-nm repeating structure of B-tubule from mouse sperm flagella
Method: EM (subtomogram averaging) / Resolution: 6.7 Å

EMDB-41317: CryoET reconstruction of 48-nm repeat doublet microtubule from human sperm
Method: EM (subtomogram averaging) / Resolution: 10.4 Å

EMDB-41320: 48 nm-repeating structure of doublets from Tektin5-KO mouse sperm axoneme
Method: EM (subtomogram averaging) / Resolution: 9.4 Å

EMDB-41431, PDB-8to0:
48-nm repeating structure of doublets from mouse sperm flagella
Method: EM (subtomogram averaging) / Resolution: 7.7 Å

EMDB-41450: Focused reconstruction of mouse doublet (register #1)
Method: EM (subtomogram averaging) / Resolution: 7.7 Å

EMDB-41451: Focused reconstruction of doublets from mouse sperm (register #2)
Method: EM (subtomogram averaging) / Resolution: 7.7 Å

Source
  • mus musculus (house mouse)
KeywordsSTRUCTURAL PROTEIN / Mammalian sperm / axoneme / microtubule-based structure / microtubule inner protein / non-motor proteins / cellular motility / fertility

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