HT structure determination of GPCRs by LCP serial femtosecond nanocrystallography

LCP 系列飞秒纳米晶体学测定 GPCR 的 HT 结构

• 批准号: 8612932
• 负责人: Vadim Cherezov
• 金额: $ 5万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8612932
• 关键词: Address; Arizona; Behavior; Cathepsins B; Cells; Collaborations; Complex; Data; Data Collection; Data Set; Deposition; Detergents; Development; Digestion; Drug Targeting; Electrons; Environment; Family; Freezing; Funding; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Gel; Generations; Goals; HIV; Harvest; Human; Human Genome; Immune; Laboratories; Lasers; Lead; Ligands; Malignant Neoplasms; Membrane; Membrane Proteins; Methods; Muramidase; Pathway interactions; Phase; Photosystem I; Physiologic pulse; Physiological; Porifera; Process; Production; Proteins; Protocols documentation; Psyche structure; Radiation; Reaction; Reproduction; Research; Resolution; Roentgen Rays; Running; Sampling; Scientist; Signal Transduction; Solid; Solutions; Solvents; Source; Stream; Structure; Suby' s G solution; Support System; Synchrotrons; Technology; Testing; Therapeutic; Time; Universities; Validation; Vision; base; beamline; computerized data processing; density; drug discovery; drug market; extracellular; improved; member; mimetics; novel; pathogen; programs; public health relevance; receptor; response; silicon nitride; software development; success; three dimensional structure

This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipeline for the atomic-level structural characterization of membrane protein microcrystals grown in membrane-like environment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electron X-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals, effectively eliminate radiation damage issues through "diffraction before destruction" (i.e., diffraction data are collected prior to onset of any damage), simplify handling, as harvesting and freezing are not required, and significantly reduce the time from obtaining initial crystal hits to collecting full data sets. Our long term goal is the integration of this technology into our structural determination pipeline enabling the determination of a large number of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressing questions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Our goal will be achieved through the following specific aims. Aim 1: Develop protocols for the production of samples of GPCR-ligand complexes and for the generation, and characterization of large number of microcrystals that can be used for SFX studies. Aim 2: Develop protocols for SFX data collection, processing and structure solution of GPCR-ligand complexes. Aim 3: Integrate protocols developed in Aims 1 and 2 into the GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining the structure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largest family of membrane proteins in the human genome with approximately 800 members and are responsible for transmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses, including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; several GPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multiple effector pathways has profound therapeutic implications, which underscores the need to understand the receptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currently marketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limited by a lack of structural information. The structure determination of GPCRs is hampered by the difficulty of preparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high- resolution structure determination even when using state-of-the art microfocus beamlines at synchrotron sources.

该项目旨在开发协议，从而建立强大的高通量管道 用于在类膜中生长的膜蛋白微晶的原子级结构表征 使用自由电子连续飞秒纳米晶体学 (SFX) 的脂质立方相 (LCP) 环境 X 射线激光源 (XFEL)。通过使用SFX，我们将不再需要获得大晶体， 通过“破坏前的衍射”有效消除辐射损伤问题（即衍射数据 在发生任何损害之前收集），简化处理，因为不需要收获和冷冻，并且 显着缩短从获得初始晶体命中到收集完整数据集的时间。我们的长期目标是 将该技术集成到我们的结构测定管道中，可以测定大量的 G 蛋白偶联受体 (GPCR)-配体复合物的三维结构数量 关于使用基于结构的药物发现（SBDD）方法的配体选择性和功效的问题。我们的 目标将通过以下具体目标来实现。目标 1：制定生产协议 GPCR-配体复合物的样品以及大量的生成和表征 可用于 SFX 研究的微晶体。目标 2：开发 SFX 数据收集、处理协议 GPCR-配体复合物的结构解析。目标 3：将目标 1 和 2 中开发的协议集成到 GPCR 结构确定管道，并通过确定以下参数来优化和验证修改后的管道 新型 GPCR 的结构，包括许多受体-配体复合物。 GPCR 构成了最大的 人类基因组中的膜蛋白家族，约有 800 个成员，负责 在细胞内传递各种细胞外信号，从而控制所有主要的生理反应， 包括视觉、嗅觉、免疫防御、生殖、消化、心理行为等；一些 GPCR 被用作 HIV 和其他病原体进入的辅助受体。 GPCR 信号通过多个 效应通路具有深远的治疗意义，这强调了了解效应通路的必要性 受体的生化和结构在适当的背景下。 GPCR 是目前约 40% 的药物的目标 已上市的药品。然而，对其作用机制和配体选择性的详细了解是有限的 由于缺乏结构信息。 GPCR 的结构测定因以下困难而受到阻碍： 制备大量均质且稳定的样品并生长足够大的晶体以实现高 即使在同步加速器上使用最先进的微焦点光束线，也能确定分辨率结构 来源。