Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Department of Chemistry, University of Oxford, Oxford, UK.
Nat Methods. 2021 Jan;18(1):69-75. doi: 10.1038/s41592-020-01021-2. Epub 2021 Jan 6.
Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.
单颗粒冷冻电子显微镜（cryo-EM）技术已成为结构生物学领域的强大工具。然而，无法可靠地制备纯均一的膜蛋白样品，阻碍了其结构测定的进展。在这里，我们开发了一种自下而上的迭代方法 Build and Retrieve (BaR)，该方法可从异质不纯的蛋白质样品中鉴定和确定各种内外膜蛋白，包括不同大小和维度的膜蛋白复合物的 cryo-EM 结构。我们还使用 BaR 方法从大肠杆菌 K12 粗膜和原始裂解物中阐明结构信息。研究结果表明，有可能从单个异质样品中解决许多相对较小（<100 kDa）和丰度较低（<10%）的未鉴定膜蛋白的高分辨率结构。重要的是，这些结果突出了 cryo-EM 在系统结构蛋白质组学中的潜力。
