A microED pipeline for the pharmaceutical and biotechnology industry

项目摘要/摘要 微晶电子衍射是一种新兴的结构测定技术，具有广泛的应用前景 制药和生物技术行业。微电子能谱能快速确定原子分辨结构 从微晶体中提取，样品要求最低。此第二阶段提案的目标是开发和交付 为商业部门确定微ED结构提供强大的服务。对关键的改进 此工作流中的步骤将支持对任何合适的样本类型进行分析，同时提供以下效率级别 符合基于结构的药物设计(SBDD)和药物化学计划的要求。这个 可允许的样品要求、最短的时间要求和生成的高质量结构 MicroED是一种吸引人的结构测定技术，在小分子、多肽、 天然产物、蛋白质和蛋白质-药物复合体。一条高效的微ED管道将有助于降低成本 缩短药物进入临床所需的时间。 MicroED使用透射式电子显微镜(TEM)从小晶体中收集衍射数据 样本。所需步骤包括将晶体样品转移到透射电子显微镜网格；收集一系列 来自单晶的连续旋转电子衍射数据集；处理衍射数据和精炼 原子模型。最初的开发阶段展示了高质量的衍射数据集 对于20多个样本，以及提供适合于满足以下需求的原子分辨率图的能力 我们的制药客户参与了阿尔法和贝塔测试。将开发新的方法，现有的 优化的方法以改进此管道的每一步，大幅增加吞吐量，并改进最终 地图和模型。具体目标包括：(1)优化瞬变电磁网格准备，确保传输足够的 在网格衬底上放置多个高质量的目标微晶体，而不损坏它们或改变它们 属性。(2)开发自动化、高通量数据收集系统，能够无人值守， 通宵采集多个不同样本的数据，并提供信息以通知数据缩放和 测定手性小分子的手性。(3)自动化和改进数据处理、分阶段和 模型改进包括实时数据缩减、动态强制阶段划分和广泛的调查 一系列新的方案和方法。 在第二阶段研究计划的同时，提供全面的商业服务，涵盖 将开发微型ED的能力。

Project Summary/Abstract Microcrystal electron diffraction (microED) is an emerging structure determination technique of high interest to the pharmaceutical and biotechnology industries. MicroED can rapidly determine atomic-resolution structures from microcrystals with minimal sample requirements. The goal of this Phase II proposal is to develop and deliver robust service offerings for microED structure determination to the commercial sector. Improvements to critical steps in this workflow will enable analysis for any suitable sample type while offering a level of efficiency that meets the requirements of Structure-Based Drug Design (SBDD) and medicinal chemistry programs. The permissive sample requirements, minimal time requirements, and high-quality structures generated make microED an attractive structure determination technique with broad applications for small molecules, peptides, natural products, proteins and protein-drug complexes. An efficient microED pipeline will help to reduce the cost of drug development and reduce the time it takes a drug to make it into the clinic. MicroED uses a transmission electron microscope (TEM) to collect diffraction data from small crystalline samples. The steps required include transferring crystalline samples to TEM grids; collecting a series of continuous-rotation, electron-diffraction datasets from single crystals; processing the diffraction data and refining the atomic model. An initial development phase demonstrated collection of excellent quality diffraction datasets for more than 20 samples, and the ability to deliver atomic resolution maps suitable for addressing the needs of our pharmaceutical clients who took part in alpha and beta testing. New methods will be developed, and existing approaches optimized to improve every step of this pipeline, substantially increase throughput, and improve final maps and models. Specific aims include: (1) Optimizing TEM grid preparation to ensure the transfer of a sufficient number of high-quality target microcrystals onto the grid substrate without damaging them or altering their properties. (2) Developing an automated, high-throughput data collection system capable of unattended, overnight data acquisition for multiple different samples, and of providing information to inform data scaling and determining handedness of chiral small molecules. (3) Automating and improving data processing, phasing and model refinement including real-time data reduction, brute force on-the-fly phasing, and investigating a wide range of new programs and methods. In parallel with the Phase II research plan, a full commercial service offering spanning the complete range of microED capabilities will be developed.