Transcontinental EM Initiative for Membrane Protein Structure

跨大陆 EM 膜蛋白结构倡议

• 批准号: 8500378
• 负责人: David L. Stokes
• 金额: $ 162.37万
• 依托单位: NEW YORK STRUCTURAL BIOLOGY CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500378
• 关键词: Address; Adsorption; Antibodies; Area; Binding; Biological; Biological Process; Biology; Cell Adhesion; Cell Adhesion Molecules; Cell physiology; Cells; Chemicals; Collaborations; Complex; Computational algorithm; Cryoelectron Microscopy; Crystallization; Crystallography; Data; Data Collection; Databases; Detergents; Development; Dialysis procedure; Disease; Drug Design; Electron Microscopy; Environment; Enzymes; Escherichia coli; Evaluation; Excision; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genomics; Hand; Health; Heme; Human; Human Biology; Image; Image Analysis; Individual; Insecta; Ion Pumps; Joints; Ligands; Lipids; Maps; Mediating; Membrane; Membrane Proteins; Methods; Micelles; Modeling; Molecular; Molecular Models; Morphology; Participant; Pattern; Peptide Hydrolases; Pharmaceutical Preparations; Play; Process; Production; Prokaryotic Cells; Protein Structure Initiative; Proteins; Pump; Research Infrastructure; Research Personnel; Resolution; Robotics; Roentgen Rays; Role; Sampling; Scheme; Signal Transduction; Software Tools; Staining method; Stains; Structural Protein; Structure; System; TRP channel; Technology; Transmembrane Transport; Work; X-Ray Crystallography; apical membrane; aquaporin-2; base; data exchange; electron crystallography; experience; graphical user interface; human PHEMX protein; human disease; image processing; innovation; interest; lens; molecular modeling; multi drug transporter; novel; novel strategies; particle; protein complex; protein expression; protein structure; receptor; reconstruction; scale up; screening; secretase; software development; tool; trafficking; two-dimensional; water channel

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.

生物膜围绕所有细胞，并介导其与外界的所有相互作用。根据生物学环境，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，因此有助于多种必需的细胞功能。我们建议建立膜蛋白结构的跨大陆EM倡议作为PSI：膜蛋白结构测定的生物学中心。根据参与者及其合作者的生物学利益，我们选择了一组主要是真核靶标，它们在人类生物学和疾病中起着重要作用。特别是，我们的靶标参与膜运输（Aquaporin，TRP和Girk通道，离子泵，用于药物和血红素的转运蛋白），在信号传导（G蛋白夫妇受体，膜内蛋白酶和细菌两组分组系统）以及细胞粘附中（Aquaporin and Mp20 Tetraspanin the Lens）。这些目标中的许多靶标在膜结合的亚基或可溶性伙伴之间形成复合物，因此对常规结构确定方法（即X射线和NMR）表示了重大挑战。我们将使用冷冻电子显微镜（Cryo-EM）作为结构确定的工具，主要是通过在脂质双层内生长二维晶体，​​还通过对洗涤剂胶束中的孤立复合物进行成像。确实，Cryo-EM在原子分辨率下确定结构确定方面已建立了记录，并通过提供更少的结晶约束和天然膜环境来为膜蛋白提供优势。对于我们的中心，我们汇集了四位在电子晶体学方面具有丰富经验的研究者，以建立用于筛选和优化2D结晶的高通量方法。第五个研究者在计算冷冻EM中具有很强的往绩，并将带头努力开发结构确定的新方法。我们坚信，通过将高通量方法应用于2D结晶，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。