X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

中文翻译：

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自下而上的结构蛋白质组学：从细胞环境中富集的蛋白质复合物的冷冻电镜。

X 射线晶体学通常需要涉及突变或截断的非天然构建体，并且受到膜蛋白和大型多组分复合物的挑战。我们在这里提出了一种自下而上的内源性结构蛋白质组学方法，其中近原子分辨率的冷冻电子显微镜 (cryoEM) 图从直接从内源性细胞环境中富集的不明蛋白质复合物从头开始重建，然后是蛋白质的鉴定和原子建模。每个复合物中的蛋白质都使用 cryoID 进行鉴定，这是我们开发的一种程序，用于从 ab initio cryoEM 图中鉴定蛋白质。作为原理证明，我们将这种方法应用于引起疟疾的寄生虫恶性疟原虫，这是一种抵抗传统结构生物学方法的生物，获得与寄生虫红细胞内存活有关的多种蛋白质复合物的原子模型。我们的方法广泛适用于确定直接从内源性来源富集的未发现蛋白质复合物的结构。