Atomic Level Analysis of Biomolecular Structure

生物分子结构的原子水平分析 该提案的总体目标是开发用于分析结构的增强方法， 生物大分子在原子水平的分辨率。我们在最近的成就基础上 和世界范围内的经验表明，单波长和多波长异常衍射（SAD和 MAD）现在主要用于从头测定大分子晶体结构。我们还建立 关于我们自己在高分辨率结构测定方面的努力的演变， 低温电子显微镜（cryo-EM）。我们的理念是通过以下方式推动方法的发展： 对当前具有生物学意义的问题的迫切应用的需求。我们建议 优化SAD和MAD定相程序，并加强生物分子的冷冻EM分析 在膜蛋白、大分子聚集体、真核生物等研究过程中， 蛋白质和其他挑战。总体目标体现在三个具体目标：（1）我们 建议加强异常衍射定相程序的挑战性问题。一个焦点被 通过组合数据提高异常衍射信噪比的改进方法 许多水晶。第二个重点是增强MAD程序，以实现准确的实验阶段 评价(2)我们建议开发的操纵和衍射分析的方法， 微晶其中一个焦点是内在的小晶体，如生长在细胞膜中的膜蛋白。 第二个焦点是用于使吸收最小化的微晶， 如低能量原生SAD实验所需，或用于时间分辨的快速扩散 实验(3)我们建议使用经验来增强分析低温EM结构的方法 在分析x射线晶体结构和验证原子模型解释的拟合中获得 实验电磁图

Atomic Level Analysis of Biomolecular Structure The overall objective of this proposal is to develop enhanced methods for analyzing structures of biological macromolecules at an atomic level of resolution. We build on our recent accomplishments and worldwide experience showing that single- and multi-wavelength anomalous diffraction (SAD and MAD) now predominate for de novo determinations of macromolecular crystal structures. We also build on the evolution of our own efforts in high-resolution structure determination to include analyses by cryogenic electron microscopy (cryo-EM). Our philosophy is to drive the development of methods by the demands of compelling applications to current problems of biological significance. We propose to optimize SAD and MAD phasing procedures and to enhance cryo-EM analyses of biomolecules at the atomic level in the course of studies of membrane proteins, macromolecular assemblages, eukaryotic proteins and other challenges. The overall objective is embodied in three specific aims: (1) We propose to enhance anomalous diffraction phasing procedures for challenging problems. One focus is on improved methods for increasing signal-to-noise ratios for anomalous diffraction by combining data from many crystals. A second focus is on enhanced MAD procedures for accurate experimental phase evaluation. (2) We propose to develop methods for the manipulation and diffraction analysis of microcrystals. One focus is on intrinsically small crystals, such as those of membrane proteins grown in lipidic cubic phase; and a second focus is on microcrystals purposed for the minimization of absorption, as is needed for low-energy native SAD experiments, or for rapid diffusion in time-resolved experiments. (3) We propose to enhance methods for analyzing cryo-EM structures using experience gained in analyzing x-ray crystal structures and in validating the fittings of atomic model interpretations to experimental EM maps.