Multimodal cryoEM approaches enable reaching sub-nanometre resolutions in situ and modelling of known components in a high-torque flagellar motor

多模式冷冻电镜方法能够在原位达到亚纳米分辨率，并对高扭矩鞭毛电机中的已知组件进行建模

• 批准号: 2131359
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2131359
• 关键词: Multimodal; cryoEM; approaches; enable; reaching

Understanding how evolutionary mechanisms gave rise to contemporary cellular pathways and protein complexes is key in understanding the development of life on earth. However, as there is no fossil record to provide glimpses of their ancestral forms, we are limited to deducing ancestral intermediates from observing contemporary variation.The bacterial flagellar motor is a great case study for understanding evolution of macromolecular complexes. It enables swimming by rotating a flagellar filament and its mechanistic core is conserved across all bacteria. However, different bacterial clades evolved divergent higher-torque motors by incorporating additional protein components on top of the conserved core. This high degree of variation makes the flagellar motor well-suited for studying how new proteins are incorporated into existing macromolecular machines.Campylobacter jejuni and related epsilon-proteobacteria have complex high-torque motors and will serve as models for me to study evolution on a molecular scale. I am initially focussing on two epsilon-proteobacterial proteins, PflA and PflB, building on previous work from our lab. PflAB form periplasmic disk structures, thought to function as scaffolding by interacting with other motor components, recruited by an ancestor of epsilon-proteobacteria.Where did additional proteins, such as PflAB, come from and how did they initially associate with the motor? What changes enabled them to be integral to the motor? What is the order of incorporation of additional components? My approach to tackle these questions is threefold.Firstly, in situ cryo electron-tomography and subtomogram averaging will yield low-to-intermediate resolution contextual information. Imaging bacterial mutants with a deleted protein of interest helps determine its location within the motor. Earlier work by collaborators identified two proteins, essential for C. jejuni motility. To help understand their functions and determine if they are part of the periplasmic scaffold, I will image their deletion mutants. Imaging other relevant bacteria will help build the motors evolutionary history. Bdellovibrio bacteriovorus is a related bacterium with a potential distant PflA homolog. Determinig the motor structure of this deletion mutant will help establish if the protein is a PflA homolog, and whether the motor is descended from an early precursor before incorporation of other accessory proteins.Secondly, in vitro work will provide higher resolution information on protein domains and atomic structure. To become part of a complex, a protein must evolve a binding interface. To define protein binding interfaces between periplasmic flagellar proteins (e.g. PflAB, MotB), I will perform pull-down assays for protein pairs, believed to interact, using different truncations to identify interacting regions. Additionally, I will attempt to obtain atomic structures of purified proteins and protein complexes by combination of X-ray crystallography and single particle analysis.Finally, in silico phylogenetics and sequence analysis will complement wet lab research. To build an evolutionary history of epsilon-proteobacterial motors and help determine order of recruitment of peripheral proteins, we will build a phylogeny of accessory proteins and compare with phylogeny of core flagellar components. PflAB contain many tandem TPR motifs, repeats often present in protein-binding interfaces. We will tpredict PflAB protein-binding regions, comparing sequences between motifs and from dn/ds ratios (rates of non-synonymous and synonymous mutations), which will enhance our understanding of the functional mechanisms of PflAB in the motor.These sets of structural, bioinformatic, phylogenetic, and molecular biological experiments will be highly informative about the role of these proteins in the flagellar motor, how they perform these functions, their evolutionary mechanisms and path to incorporation, contributing to our

理解进化机制如何产生当代细胞途径和蛋白质复合物是理解地球生命发展的关键。然而，由于没有化石记录来提供它们祖先形式的一瞥，我们仅限于从观察当代变异中推断祖先中间体。细菌鞭毛马达是理解大分子复合物进化的一个很好的案例研究。它通过旋转鞭毛丝使游泳成为可能，其机械核心在所有细菌中都是保守的。然而，不同的细菌进化枝通过在保守的核心之上结合额外的蛋白质组分来进化出不同的高扭矩马达。这种高度的变异使得鞭毛马达非常适合研究新蛋白质如何被整合到现有的大分子机器中。空肠弯曲杆菌和相关的ε-变形菌具有复杂的高扭矩马达，将作为我在分子尺度上研究进化的模型。我最初专注于两个epsilon-proteobacterial蛋白质，PflA和PflB，建立在我们实验室以前的工作。PflAB形成周质盘结构，被认为是通过与其他马达组件相互作用而起支架作用，由ε-变形菌的祖先招募。额外的蛋白质，如PflAB，来自哪里？它们最初是如何与马达相关联的？是什么变化使它们成为发动机的组成部分？添加其他组件的顺序是什么？我解决这些问题的方法有三个：首先，原位低温电子断层扫描和亚断层图像平均将产生低到中等分辨率的上下文信息。对具有缺失的感兴趣蛋白质的细菌突变体进行成像有助于确定其在马达内的位置。合作者的早期工作确定了两种蛋白质，对C。空肠运动为了帮助理解它们的功能并确定它们是否是周质支架的一部分，我将对它们的缺失突变体进行成像。对其他相关细菌进行成像将有助于建立马达的进化历史。噬菌蛭弧菌是一种具有潜在远缘PflA同源物的相关细菌。确定这种缺失突变体的马达结构将有助于确定该蛋白是否为PflA同源物，以及马达是否起源于其他辅助蛋白掺入之前的早期前体。要成为复合物的一部分，蛋白质必须进化出一个结合界面。为了定义周质鞭毛蛋白（例如PflAB，MotB）之间的蛋白质结合界面，我将使用不同的截短来鉴定相互作用区域，对被认为相互作用的蛋白质对进行下拉测定。此外，我将尝试通过X射线晶体学和单粒子分析相结合来获得纯化蛋白质和蛋白质复合物的原子结构。最后，计算机遗传学和序列分析将补充湿实验室研究。为了建立ε-proteobacterial马达的进化历史，并帮助确定外周蛋白质的招募顺序，我们将建立辅助蛋白质的同源性，并与核心鞭毛组件的同源性进行比较。PflAB含有许多串联的TPR基序，重复序列通常存在于蛋白结合界面。我们将通过比较基序之间的序列和dn/ds比值来预测PflAB蛋白结合区这些结构、生物信息学、系统发育和分子生物学实验的集合将是关于这些蛋白质在鞭毛马达中的作用、它们如何执行这些功能、它们的进化机制和合并途径，有助于我们