Preparing for a warmer future: Conformational ensembles, dynamics and interactions from variable temperature crystallography

了解蛋白质和其他生命分子的3D结构对于理解它们的功能和设计新药至关重要。大多数原子分辨率的蛋白质结构是使用X射线晶体学确定的。 但是，虽然蛋白质在生物温度（通常接近室温）下发挥作用，但在过去的十年中，几乎所有的X射线晶体学都是在冷却到低温的蛋白质晶体上进行的，其中许多重要的结构和动力学细节可能会受到影响。这个项目将探索丰富的额外信息，可能是使用蛋白质晶体与液体溶剂访问。 在~330和200 K之间的温度下收集的结构数据集将允许探索蛋白质构象集合和能量景观如何演变，以及导致冷变性的签名和相互作用。超快和可变速率冷却将用于表征大型和/或高度相关的蛋白质运动的时间尺度，并更好地保留室温结构以供在100 K下进行检查。该项目以最近在样品处理和数据收集以及改进和分析工具方面的发展为基础，并旨在大幅度推广这些发展。 该项目将扩大蛋白质晶体学研究的范围，并有助于全面了解蛋白质结构和功能之间的关系，这与理解生物学和开发新的生物活性化合物有关。 它建立在过去基础研究和方法开发的基础上，这些基础研究和方法开发导致了世界各地晶体学家使用的商业工具。本科生和研究生研究人员将接受先进方法的跨学科培训，并将这些方法传播到更广泛的结构生物学社区至关重要。本研究计划的补充，在本科导论课程改革，旨在提高生命科学与工程专业学生的保留和学科掌握的大力努力。 它还辅之以STEM教师招聘和培训以及机构变革方面的广泛努力，为以研究为重点的STEM部门如何解决我们初中和高中教师短缺问题提供了一个模式。

An understanding of the 3D structure of proteins and other molecules of life is essential to understanding their function and to the design of new medicines. Most atomic-resolution protein structures are determined using X-ray crystallography. But while proteins function at biological (generally, near room) temperature, in the last decade nearly all X-ray crystallography has been performed on protein crystals cooled to cryogenic temperatures, where many important details of structure and dynamics may be compromised. This project will explore the wealth of additional information that may be accessible using protein crystals with liquid solvent. Structural data sets collected at temperatures between ~330 and 200 K will allow exploration of how protein conformational ensembles and energy landscapes evolve, and of the signatures of and interactions leading to cold denaturation. Ultra-fast and variable-rate cooling will be used to characterize timescales of large and/or highly correlated protein motions, and to better preserve room temperature structure for examination at 100 K. The project builds on recent developments in sample handling and data collection and in refinement and analysis tools, and aims to substantially extend these developments. Broader Impacts This project will enable a broadening in the scope of crystallographic studies of proteins, and contribute to a comprehensive understanding of the relation between protein structure and function, relevant to the understanding biology and to the development of new biologically active compounds. It builds on past efforts in fundamental studies and methods development that have led to commercial tools used by crystallographers worldwide. Undergraduate and graduate student researchers will receive cross-disciplinary training in advanced methods and will be critical in propagating these methods to the broader structural biology community. This research program is complemented by vigorous efforts in undergraduate introductory course curriculum reform aimed at improving the retention and subject mastery of life science and engineering students. It is also complemented by a broad effort in STEM teacher recruiting and training and in institutional change that provides a model for how research-focused STEM departments can address critical teacher shortages in our middle and high schools.