TRD2: Phasing and refinement

TRD2：分阶段和细化

• 批准号: 10155528
• 负责人: PAWEL A. PENCZEK
• 金额: $ 24.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10155528
• 关键词: Acceleration; Adopted; Biological; Biological Assay; Computer software; Crystallization; Data; Data Analyses; Data Collection; Development; Diffusion; Dose Fractionation; Electron Beam; Electron Diffraction Microscopy; Electron Microscopy; Electrons; Exposure to; Fourier Transform; Frequencies; Heavy Metals; Homologous Gene; Image; Ions; Lead; Methodology; Methods; Modality; Modeling; Modification; Molecular; Pattern; Phase; Physics; Procedures; Process; Proteins; Radiation induced damage; Reagent; Resolution; Roentgen Rays; Sampling; Signal Transduction; Spottings; Statistical Distributions; Statistical Methods; Structure; System; Technology; Work; X-Ray Crystallography; absorption; base; computerized data processing; detector; electron diffraction; experimental study; foot; microscopic imaging; nanocrystal; protein structure; small molecule; statistics; tool; voltage

TRD 2. Phasing and refinement - Penczek (Lead) Summary Similarly as X-ray crystallography, microcrystal electron diffraction (MicroED) delivers amplitudes of the imaged object Fourier transform and the phase information is lost. Several procedures have been developed in X-ray crystallography for solving the phase problem for de novo structure determination. These include Patterson difference, molecular replacement, ab initio statistical methods, heavy metal (multi- and single isomorphous replacement), damage-based phasing, and anomalous dispersion. We already successfully applied MicroED to the determination of new protein structures using two approaches: (1) direct ab initio and molecular replacement using idealized models and (2) molecular replacement using homologues. However, ab initio methods are feasible only in cases when the obtained resolution is better than ~1.2Å while molecular replacement is only possible if the new protein is homologous with another already known structure. Other X-ray methods are either not applicable to MicroED (for example anomalous dispersion) or were not yet adapted and implemented for MicroED. Here we will expand the pallet of available tools by developing dedicated MicroED phasing methods for routine de novo structure determination in cases in which the diffraction spots do not reach spatial frequencies required by ab initio methods and molecular replacement is not possible due to lack of homologous structural information. To facilitate phasing, we will develop comprehensive MicroED data scaling and integration methodologies taking advantage of maximum likelihood approaches to deliver accurate intensity values. For de novo phasing we will adapt two methods that were historically most successful in X-ray crystallography: (1) heavy metal isomorphous replacement strategies and (2) phasing by specific radiation damage. The aims are: 1. Development of a comprehensive MicroED data scaling and integration methodology; 2. De novo phasing using isomorphous replacement; 3. De novo phasing using radiation damage. The successful completion of the Aims listed will bring MicroED to an equal footing with X-ray crystallography. In the long-run, the proposed developments will lead to establishment of MicroED as a method capable of phasing and solving structures of entirely new and biologically important systems that currently are not tractable by X-ray crystallography.

TRD2。相平进和精炼-Penczek（铅） 概括 与X射线晶体学类似，微晶电子衍射（微晶体）提供成像的放大器 对象傅立叶变换，阶段信息丢失。 X射线已经开发了几个程序 用于解决从头结构确定的相位问题的晶体学。这些包括帕特森 差异，分子置换，从头开始统计方法，重金属（多形和单形的） 替换），基于损伤的相位和异常分散。我们已经成功地将其应用于 使用两种方法确定新蛋白质结构：（1）直接依次和分子替代 使用理想化的模型和（2）使用同源物的分子替代。但是，从头开始方法是 仅在获得的分辨率优于〜1.2Å的情况下，可行 如果新蛋白质与另一种已知的结构同源，则可能。其他X射线方法是 不适用于微型（例如异常分散）或尚未适应和实施 微型。在这里，我们将通过开发专用的微型相位方法来扩展可用工具的托盘 对于衍射点未达到空间频率的情况，对于常规的从头结构确定 由于缺乏同源的结构 信息。为了促进相位，我们将开发全面的微型数据扩展和集成 方法利用最大似然方法来传递准确的强度值。为de Novo逐步逐步我们将适应两种历史上最成功的X射线晶体学的方法：（1）重型 金属同构替换策略和（2）通过特定的辐射损伤进行逐步估计。目的是：1。 开发全面的微型数据扩展和集成方法； 2。使用 异阵代替； 3。使用辐射损伤从头逐渐缩小。成功完成目标 列出的将使微型与X射线晶体学相等地基础。从长远来看，提议 发展将导致建立微型，作为一种能够相相和解决结构的方法 X射线晶体学目前无法处理的全新和生物学上重要的系统。