Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes ex vivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila.

冷冻电子显微镜 (cryo-EM) 彻底改变了大分子复合物的结构测定。随着离体和原位结构生物学向高度复杂的内源性大分子复合物结构测定的范式转变，天然复合物的结构数量越来越多。这些复合物通常含有与不同细胞状态或过程相关的未识别蛋白质。以低于 4 Å 的分辨率识别蛋白质仍然具有挑战性，因为侧链无法可靠地可视化。在这里，我们推出了 DomainFit，这是一个从冷冻电镜图谱中进行半自动域级蛋白质识别的程序，特别是在分辨率低于 4 Å 的情况下。通过将 AlphaFold2 预测模型中的域拟合到冷冻电镜图中，该程序执行统计分析并尝试识别形成密度的域和候选蛋白质。使用 DomainFit，我们鉴定了两种微管内部蛋白，其中一种包含 CCDC81 结构域，并且专门位于嗜热四膜虫双联微管的近端区域。