TRD2: Phasing and refinement

TRD2：分阶段和细化

• 批准号: 10460923
• 负责人: PAWEL A. PENCZEK
• 金额: $ 24.47万
• 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/10460923
• 关键词: Acceleration; Adopted; 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; imaging facilities; 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.

TRD 2. 分阶段和完善 - Penczek（领导） 概括 与 X 射线晶体学类似，微晶电子衍射 (MicroED) 可提供成像的振幅 物体傅立叶变换和相位信息丢失已经在X射线中开发了几种程序。 用于解决从头结构确定的相位问题的晶体学包括帕特森。 差异、分子置换、从头算统计方法、重金属（多同晶和单同晶） 替换）、基于损伤的定相和异常色散我们已经成功地将 MicroED 应用于。 使用两种方法确定新的蛋白质结构：(1) 直接从头开始和分子替换 使用理想化模型和（2）使用同系物进行分子替换然而，从头开始方法是。 仅当获得的分辨率优于 ~1.2Å 时才可行，而分子替换仅适用于 如果新蛋白质与另一种已知结构同源，则可能有其他 X 射线方法。 不适用于 MicroED（例如反常色散）或尚未适应和实施 在这里，我们将通过开发专用的 MicroED 定相方法来扩展可用工具的范围。 用于衍射斑未达到空间频率的情况下的常规从头结构测定 由于缺乏同源结构，从头算方法所需的分子替换是不可能的 为了促进分阶段，我们将开发全面的 MicroED 数据扩展和集成。 利用最大似然方法来提供准确的强度值的方法。 在 novo 定相中，我们将采用两种历史上最成功的 X 射线晶体学方法：(1) 重 金属同晶替代策略和（2）按特定辐射损伤分阶段目标是：1。 开发全面的 MicroED 数据扩展和集成方法；2. 使用 De novo 定相； 同晶置换；3.利用辐射损伤从头定相成功完成了目标。 从长远来看，该技术的上市将使 MicroED 与 X 射线晶体学处于同等地位。 将导致 MicroED 的建立，作为一种能够开发分阶段和解决结构的方法 全新且具有重要生物学意义的系统目前无法通过 X 射线晶体学来处理。