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.

该项目旨在制定协议，以建立一个强大的高通量管道 用于在膜样生长中生长的膜蛋白微晶的原子级结构表征 自由电子飞秒纳米微阵列（SFX）技术在立方相（LCP）环境中的应用 X射线激光源（XFEL）。随着SFX的使用，我们将不再需要获得大晶体， 通过“破坏前的衍射”（即，衍射数据 在任何损害发生之前收集），简化处理，因为不需要收获和冷冻，以及 显著减少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， G蛋白偶联受体（GPCR）-配体复合物的三维结构的数量， 使用基于结构的药物发现（SBDD）方法的配体选择性和有效性问题。我们 将通过以下具体目标实现这一目标。目标1：制定生产 GPCR-配体复合物的样品，并用于产生和表征大量的 可用于SFX研究的微晶。目标2：制定SFX数据收集、处理和 以及GPCR-配体复合物的结构解析。目标3：将目标1和2中制定的协议纳入 GPCR结构确定流水线，并通过确定 包括许多受体-配体复合物的新型GPCR的结构。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、精神行为等; GPCR被用作HIV和其他病原体进入的共受体。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 受体的生物化学和结构在适当的情况下。GPCR是目前约40%的目标 市售药品。然而，对它们的作用机制和配体选择性的详细了解是有限的 缺乏结构性信息。GPCR的结构测定受到以下困难的阻碍： 制备大量均匀和稳定的样品，并生长足够大的晶体， 即使在同步加速器上使用最先进的微聚焦光束线， 源