EAGER: Methodology development for 3D atomic-scale structural dynamics movies of enzymes

捕捉生物机器执行其功能的动态结构仍然是生物学的圣杯。传统的工具只能提供沿反应途径稳定状态的结构“快照”。这个项目旨在开发一种方法，可以捕捉到大分子结构在工作时的完整画面。所开发的研究工具将广泛适用于各种生物系统，并将以出版物、教程、会议报告等方式传播，以加速其采用。该项目的教育部分将强调单分子生物物理学和动态结构确定计算工具的使用。它将为高中生、本科生、研究生和博士后提供最先进的单分子生物物理学研究培训，并为研究生和博士后提供领导经验。具体来说，该项目将研究一个简单的细菌转录系统在启动过程中的转录泡结构，作为开发新技术概念的测试案例。细菌转录中某些中间体的存在表明该过程的共同特征存在于不同的生命领域，并反映了保守的调控机制；因此，这些发现将具有广泛的实用性。为了确定动态结构，该项目将结合实验和理论工具，包括多点单分子FRET测量，快速单分子微流体混合，分子动力学模拟和时间分辨单光子检测，以获取转录泡启动过程中的数据并进行分子建模。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Capturing the dynamic structure of a biological machine performing its function remains the holy grail of biology. Conventional tools provide only structural "snapshots" of stable states along the reaction pathway. This project aims to develop methods that will capture a full movie of a macromolecule's structure as it works. The research tools developed will be applicable to a wide variety of biological systems, and will be disseminated by means of publications, tutorials, conference presentations and the like, to accelerate their adoption. The education component of the project will emphasize the use of single molecule biophysics and computational tools for dynamic structure determination. It will provide high school, undergraduate, graduate students and postdocs training in state-of-the-art single molecule biophysics research, and offer leadership experiences for graduate students and postdocs as well.Specifically, this project will study the structure of the transcription bubble in a simple bacterial transcription system during initiation as a test case for developing new technological concepts. The existence of certain intermediates in bacterial transcription suggests common features of the process exist across the different domains of life and reflects a conserved regulatory mechanism; thus, the findings will have broad utility. For the purpose of dynamic structure determination, the project will combine experimental and theoretical tools, including multi-spot single-molecule FRET measurements, fast single-molecule microfluidic mixing, molecular dynamics simulations and time-resolved single-photon detection to acquire data on- and perform molecular modeling of- the transcription bubble during initiation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.