HT structure determination of GPCRs by LCP serial femtosecond nanocrystallography

该项目旨在开发将导致建立一条健壮的高吞吐量管道的协议 用于膜状生长的膜蛋白微晶的原子级结构表征 自由电子系列飞秒纳米晶(SFX)研究脂类立方相(LCP)的环境 X射线激光光源(XFELs)。随着SFX的使用，我们将不再需要获得大晶体， 通过“先绕射后销毁”有效消除辐射损害问题(即， 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 显著减少从获得初始晶体点击数到收集完整数据集的时间。我们的长期目标是 将这项技术集成到我们的结构确定流水线中，能够确定大型 G蛋白偶联受体(GPCRs)配基复合体的三维结构数目 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。我们的 目标将通过以下具体目标来实现。目标1：制定生产 GPCR-配基复合体的样品及其大量的生成和表征 可用于SFX研究的微晶体。目标2：制定SFX数据收集、处理的协议 GPCR型配体络合物的结构溶液。目标3：将目标1和目标2中制定的协议整合到 GPCR型结构确定管路，并通过确定改进型管路的 新型GPCRs的结构，包括许多受体-配体复合体。GPCRs是最大的 人类基因组中的膜蛋白家族，大约有800个成员，负责 在细胞内传递各种胞外信号，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； GPCRs被用作艾滋病毒和其他病原体进入的辅助受体。GPCR信号通过多个 效应器通路具有深刻的治疗意义，这强调了理解 受体在生物化学和结构上都是在适当的背景下。GPCR是目前约40%的目标 市场上销售的药品。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。 由于缺乏结构性信息。GPCRs的结构测定受到以下困难的阻碍 制备了大量均匀稳定的样品，并生长出足够大的晶体，以获得高密度的晶体。 即使在同步加速器使用最先进的微聚焦光束线时也能确定分辨率结构 消息来源。

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.