RT Journal Article SR Electronic T1 3D structure and in situ arrangements of CatSper channel in the sperm flagellum JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.06.19.448910 DO 10.1101/2021.06.19.448910 A1 Yanhe Zhao A1 Huafeng Wang A1 Caroline Wiesehoefer A1 Naman B. Shah A1 Evan Reetz A1 Jae Yeon Hwang A1 Xiaofang Huang A1 Polina V. Lishko A1 Karen M. Davies A1 Gunther Wennemuth A1 Daniela Nicastro A1 Jean-Ju Chung YR 2021 UL http://biorxiv.org/content/early/2021/06/19/2021.06.19.448910.abstract AB The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSper’s complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.Competing Interest StatementThe authors have declared no competing interest.