Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, 94158, USA.
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, 94158, USA.
Nat Commun. 2022 Jul 28;13(1):4366. doi: 10.1038/s41467-022-32125-2.
Single particle cryogenic-electron microscopy (cryo-EM) is used extensively to determine structures of activated G protein-coupled receptors (GPCRs) in complex with G proteins or arrestins. However, applying it to GPCRs without signaling proteins remains challenging because most receptors lack structural features in their soluble domains to facilitate image alignment. In GPCR crystallography, inserting a fusion protein between transmembrane helices 5 and 6 is a highly successful strategy for crystallization. Although a similar strategy has the potential to broadly facilitate cryo-EM structure determination of GPCRs alone without signaling protein, the critical determinants that make this approach successful are not yet clear. Here, we address this shortcoming by exploring different fusion protein designs, which lead to structures of antagonist bound A adenosine receptor at 3.4 Å resolution and unliganded Smoothened at 3.7 Å resolution. The fusion strategies explored here are likely applicable to cryo-EM interrogation of other GPCRs and small integral membrane proteins.
单颗粒低温电子显微镜（cryo-EM）被广泛用于确定与 G 蛋白或 arrestin 复合物结合的激活 G 蛋白偶联受体（GPCR）的结构。然而，将其应用于没有信号蛋白的 GPCR 仍然具有挑战性，因为大多数受体在其可溶性结构域中缺乏有助于图像对齐的结构特征。在 GPCR 晶体学中，在跨膜螺旋 5 和 6 之间插入融合蛋白是一种非常成功的结晶策略。尽管类似的策略有可能广泛促进没有信号蛋白的 GPCR 的 cryo-EM 结构测定，但使这种方法成功的关键决定因素尚不清楚。在这里，我们通过探索不同的融合蛋白设计来解决这一不足，这些设计导致了 3.4 Å 分辨率下拮抗剂结合的 A 腺苷受体和 3.7 Å 分辨率下未结合的 Smoothened 的结构。这里探索的融合策略可能适用于其他 GPCR 和小的整合膜蛋白的 cryo-EM 检测。
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