Transcontinental EM Initiative for Membrane Protein Structure

生物膜包裹着所有细胞，并调节它们与外界的所有相互作用。根据不同的生物学背景，膜蛋白作为受体、酶、通道、转运体、结构蛋白和细胞粘附分子，并因此促进了各种基本的细胞功能。我们建议建立跨大陆膜蛋白结构的EM倡议作为PSI：膜蛋白结构测定生物学中心。基于我们的参与者及其合作者的生物学兴趣，我们选择了一组在人类生物学和疾病中发挥重要作用的真核靶标。特别是，我们的目标涉及膜运输（水通道蛋白，TRP和GIRK通道，离子泵，药物和血红素的转运体），信号传导（g蛋白偶联受体，膜内蛋白酶和细菌双组分系统），以及细胞粘附（水通道蛋白和来自晶体的MP20四蛋白）。这些靶标中的许多在膜结合亚基之间或与可溶性伙伴形成复合物，因此对传统的结构测定方法（即x射线和核磁共振）提出了重大挑战。我们将使用低温电子显微镜（cryo-EM）作为我们的结构测定工具，主要是通过在脂质双层内生长二维晶体，但也通过对洗涤剂胶束内分离的复合物进行成像。事实上，低温电镜在原子分辨率结构测定方面有着良好的记录，并且通过提供更少的结晶限制和天然膜环境，为膜蛋白提供了优势。对于我们的中心，我们汇集了四名具有丰富电子晶体学经验的研究人员，以建立筛选和优化二维结晶的高通量方法。第五名研究者在计算低温电镜方面有很强的记录，并将率先努力开发结构测定的新方法。我们相信，通过将高通量方法应用于二维结晶和现代化的结构测定方法，冷冻电镜可以为我们对膜蛋白生物学的理解做出重大贡献。

Biological membranes surround all cells and mediate all their interactions with the outside world. Depending on the biological context, membrane proteins act as receptors, enzymes, channels, transporters, structural proteins and cell adhesion molecules and, as such, contribute to a wide variety of essential cellular functions. We propose to establish the Transcontinental EM Initiative for Membrane Protein Structure as a PSI: Biology Center for Membrane Protein Structure Determination. Based on the biological interests of our participants and their collaborators, we have selected a group of mostly eukaryotic targets that play important roles in human biology and disease. In particular, our targets are involved in membrane transport (aquaporin, TRP and GIRK channels, ion pumps, transporters for drugs and heme), in signaling (G-protein couple receptors, intramembrane proteases and bacterial two-component systems), and in cell adhesion (aquaporin and the MP20 tetraspanin from the lens). Many of these targets form complexes, either between membrane-bound subunits or with soluble partners, and therefore represent a significant challenge to conventional methods of structure determination (i.e., X-ray and NMR). We will use cryo-electron microscopy (cryo-EM) as our tool for structure determination, primarily by growing two-dimensional crystals within a lipid bi-layer but also by imaging isolated complexes within detergent micelles. Indeed, cryo-EM has an established track record in structure determination at atomic resolution and offers advantages for membrane proteins by providing fewer crystallization constraints and a native membrane environment. For our Center, we have brought together four investigators with extensive experience in electron crystallography to establish high-throughput methods for screening and optimizing 2D crystallization. A fifth investigator has a strong track record in computation cryo-EM and will spearhead efforts to develop novel methods for structure determination. We are convinced that by applying high-throughput methods to 2D crystallization and by modernizing our methods for structure determination, cryo-EM can make a substantial contribution to our understanding of membrane protein biology.