The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca²⁺ gateway into the sperm flagellum. However, the complex subunit composition of CatSper 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.

中文翻译：

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精子鞭毛中 CatSper 通道的 3D 结构和原位排列

精子钙通道 CatSper 作为进入精子鞭毛的主要 Ca ²⁺通道，在成功受精中发挥着核心作用。然而，CatSper 复杂的亚基组成阻碍了它在体外的重建和结构说明。在这里，我们应用冷冻电子断层扫描来可视化完整哺乳动物精子中天然 CatSper 通道复合物的大分子组织，并确定了另外三种与 CatSper 相关的蛋白质。重复的 CatSper 单元沿着鞭毛在四个纳米域中形成长的锯齿形行。在小鼠和人类精子中，每个 CatSper 重复序列都由一个四聚体孔复合物组成。鼠 CatSper 包含一个额外的向外定向的翼结构，连接到四聚体通道。大多数细胞外结构域在每个成孔通道上方形成一个冠层，与锯齿形屋顶相互连接。细胞内结构域将两个相邻的通道复合物连接到对角线阵列。Efcab9中这种细胞内链接的丢失^-/-精子扭曲纵向对齐的锯齿形图案并损害鞭毛运动。这项工作提供了对 CatSper 复合物组装和运输以产生纳米域的潜在机制的独特见解，并为理解 CatSper 在精子活力调节中的功能提供了长期寻求的结构基础。