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.

该项目旨在开发将导致建立强大高通量管道的协议 对于原子水平的结构表征，在膜状中生长的膜蛋白微晶的结构表征 脂质立方相（LCP）的环境，使用自由电子处的串行飞秒纳米结晶图（SFX） X射线激光源（XFELS）。使用SFX，我们将消除获得大晶体的需求， 通过“破坏前的衍射”有效地消除辐射损伤问题（即衍射数据是 在任何损坏发作之前收集），简化处理，因为不需要收获和冻结，并且 从获得初始晶体命中到收集完整数据集的时间大大减少了时间。我们的长期目标是 将该技术集成到我们的结构确定管道中，以确定大型 G蛋白偶联受体（GPCR）的三维结构的数量 使用基于结构的药物发现（SBDD）方法的有关配体选择性和疗效的问题。我们的 目标将通过以下特定目标实现。目标1：制定生产协议 GPCR配体配合物的样本和生成，以及大量的表征 可用于SFX研究的微晶。 AIM 2：为SFX数据收集开发协议，处理 和GPCR配体配合物的结构解。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的 新型GPCR的结构，包括许多受体配体复合物。 GPCR构成最大的 人类基因组中的膜蛋白家族，约有800名成员，负责 在细胞内部传输各种细胞外信号，从而控制所有主要的生理反应， 包括视力，嗅觉，免疫防御，繁殖，消化，心理行为等；一些 GPCR被用作艾滋病毒和其他病原体进入的共受体。 GPCR信号通过多个 效应子途径具有深远的治疗意义，这突显了了解 受体在适当的环境下在生化和结构上。 GPCR是当前约40％的目标 销售药物。但是，详细了解其作用机理和配体选择性是有限的 由于缺乏结构信息。 GPCR的结构确定受到了困难 准备大量同质和稳定的样品，并生长到足够大的晶体，以进行高 即使在同步器处使用最先进的微型焦点梁时，分辨率结构的确定 来源。