TRD2: Phasing and refinement

Trd 2.阶段化和精细化--Penczek(领先) 摘要 与X射线结晶学类似，微晶电子衍射(MicroED)提供了成像的幅度 目标傅里叶变换和相位信息丢失。在X射线中已经开发了几种程序 解决从头结构测定中的相问题的结晶学。其中包括帕特森 差异，分子置换，从头算统计方法，重金属(多重和单重同构 替换)、基于损害的阶段化和异常分散。我们已经成功地将MicroED应用到 用两种方法确定新的蛋白质结构：(1)直接从头算和分子置换 使用理想化模型和(2)使用同系物的分子置换。然而，从头算方法是 仅当获得的分辨率优于~1.2？而分子置换仅为 如果新的蛋白质与另一个已知的结构同源，就有可能。其他X射线方法有 不适用于MicroED(例如，异常色散)或尚未适应和实施 微型电子除颤器。在这里，我们将通过开发专用的MicroED阶段化方法来扩展可用工具的托盘 用于在衍射点未达到空间频率的情况下的常规从头结构确定 从头算方法所要求的，由于缺乏同源结构，分子置换是不可能的 信息。为了促进分阶段，我们将开发全面的MicroED数据缩放和集成 利用最大似然法提供准确的强度值的方法。对于De 我们将采用在X射线结晶学历史上最成功的两种方法：(1)重 金属同象替换策略和(2)特定辐射损伤的阶段性。目标是：1. 制定一个综合的MicroED数据标度和集成方法；2.使用 同构替换；3.利用辐射损伤进行去重相。圆满完成各项目标 上市将使MicroED与X射线结晶学处于同等地位。长远而言，建议的 发展将导致建立微ED作为一种能够分阶段和解决结构的方法 全新的、具有生物重要性的系统，目前无法通过X射线结晶学进行处理。

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.