De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.
De novo protein identification in mammalian sperm using in situ cryoelectron tomography and AlphaFold2 docking.
复制标题
使用原位冷冻电子断层扫描和 AlphaFold2 对接对哺乳动物精子进行从头蛋白质鉴定。
DOI：
10.1016/j.cell.2023.09.017
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发表时间：
2023
期刊：
影响因子：
64.5
通讯作者：
Agard,DavidA
中科院分区：
文献类型：
--
作者：
Chen,Zhen;Shiozaki,Momoko;Haas,KelseyM;Skinner,WillM;Zhao,Shumei;Guo,Caiying;Polacco,BenjaminJ;Yu,Zhiheng;Krogan,NevanJ;Lishko,PolinaV;Kaake,RobynM;Vale,RonaldD;Agard,DavidA
为了了解细胞通路的分子机制，当代工作流程通常需要多种技术来识别蛋白质，跟踪它们的定位，并在体外确定它们的结构。在这里，我们结合细胞冷冻电子断层扫描（cryo-ET）和AlphaFold 2建模来解决这些问题，并了解哺乳动物精子是如何原位构建的。我们的细胞冷冻ET和亚断层扫描平均提供了6.0倍的轴丝微管结构重建。解析良好的三级结构使我们能够无偏地将精子特异性密度与小鼠蛋白质组的21，615个AlphaFold 2预测蛋白质模型相匹配。我们确定Tektin 5，CCDC 105和SPACA 9为新型微管相关蛋白。这些蛋白质形成了广泛的相互作用网络交联的轴丝双线微管的内腔，这表明它们在调制的长丝的机械性能的作用。事实上，Tekt 5 −/−精子拥有更多的畸形鞭毛，弯曲180°。总之，我们的研究提出了一个细胞视觉蛋白质组学工作流程，并阐明了Tektin 5的体内功能。
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structuresin vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are builtin 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 thein vivofunctions of Tektin 5.