Transcontinental EM Initiative for Membrane Protein Structure

生物膜包围着所有的细胞，并调节它们与外界的所有相互作用。根据生物环境的不同，膜蛋白作为受体、酶、通道、转运体、结构蛋白和细胞黏附分子发挥作用，因此有助于发挥多种基本的细胞功能。我们建议建立跨洲EM膜蛋白结构倡议，作为PSI：膜蛋白结构测定生物学中心。基于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是真核细胞的靶点。特别是，我们的靶标涉及膜转运(水通道蛋白、Trp和GIRK通道、离子泵、药物和血红素的转运体)、信号传递(G蛋白偶联受体、膜内蛋白酶和细菌双组分系统)和细胞黏附(水通道蛋白和晶状体中的MP20Tetraspanin)。其中许多靶在膜结合的亚基之间或与可溶性伙伴之间形成络合物，因此对传统的结构确定方法(即X射线和核磁共振)构成了重大挑战。我们将使用冷冻电子显微镜(CRYO-EM)作为我们的结构确定工具，主要通过在脂质双层中生长二维晶体，但也通过在洗涤剂胶束中成像分离的络合物。事实上，Cryo-EM在原子分辨率的结构测定方面已经建立了良好的记录，并通过提供较少的结晶限制和天然的膜环境为膜蛋白提供了优势。对于我们的中心，我们汇集了四名在电子结晶学方面具有丰富经验的研究人员，以建立高通量方法来筛选和优化2D结晶。第五名研究人员在计算低温电磁方面有很好的记录，将带头努力开发新的结构确定方法。我们相信，通过将高通量方法应用到2D结晶中，并通过使我们的结构确定方法现代化，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.