Transcontinental EM Initiative for Membrane Protein Structure

生物膜包围着所有细胞，并介导它们与外界的所有相互作用。根据生物学背景，膜蛋白充当受体、酶、通道、转运蛋白、结构蛋白和细胞粘附分子，并且因此有助于多种基本细胞功能。我们建议建立跨大陆EM膜蛋白结构倡议作为PSI：膜蛋白结构测定生物中心。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主要真核靶点。具体而言，我们的靶点涉及膜转运（水通道蛋白、TRP和GIRK通道、离子泵、药物和血红素转运蛋白）、信号传导（G蛋白偶联受体、膜内蛋白酶和细菌双组分系统）和细胞粘附（水通道蛋白和来自透镜的MP20四跨膜蛋白）。这些靶标中的许多在膜结合亚基之间或与可溶性配偶体形成复合物，因此对结构测定的常规方法（即，X射线和NMR）。我们将使用冷冻电子显微镜（cryo-EM）作为我们的工具，结构测定，主要是通过生长二维晶体内的脂质双层，但也通过成像分离的复合物内的洗涤剂胶束。事实上，cryo-EM在原子分辨率的结构测定方面具有既定的记录，并通过提供更少的结晶约束和天然膜环境为膜蛋白提供优势。对于我们的中心，我们汇集了四名在电子晶体学方面具有丰富经验的研究人员，以建立筛选和优化2D结晶的高通量方法。第五名研究人员在计算cryo-EM方面有着良好的记录，并将带头努力开发新的结构测定方法。我们相信，通过将高通量方法应用于2D结晶和现代化我们的结构测定方法，cryo-EM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.