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

了解进化机制如何产生当代细胞通路和蛋白质复合物是理解地球上生命发展的关键。然而，由于没有化石记录提供它们祖先形式的一瞥，我们只能通过观察当代的变化来推断祖先的中间物种。细菌鞭毛马达是了解大分子复合物进化的一个很好的研究案例。它通过旋转鞭毛细丝来游泳，它的机械核心在所有细菌中都是保守的。然而，不同的细菌分支通过在保守核心上加入额外的蛋白质成分，进化出了不同的高扭矩马达。这种高度的变异使得鞭毛马达非常适合于研究新蛋白质如何与现有的大分子机器结合。空肠弯曲杆菌（Campylobacter jejuni）和相关的epsilon-proteobacteria具有复杂的高扭矩马达，将成为我在分子尺度上研究进化的模型。我最初专注于两种epsilon- proteobacterian蛋白，PflA和PflB，建立在我们实验室之前的工作基础上。PflAB形成质周盘状结构，被认为是通过与其他运动成分相互作用而起支架作用，由epsilon-proteobacteria的祖先招募。额外的蛋白质，如PflAB，从何而来？它们最初是如何与马达联系在一起的？是什么变化使它们成为发动机的一部分？附加组件的合并顺序是什么？我解决这些问题的方法有三个方面。首先，原位冷冻电子断层扫描和亚层析成像平均将产生低至中等分辨率的上下文信息。对缺失感兴趣蛋白的细菌突变体进行成像有助于确定其在马达中的位置。合作者早期的工作发现了两种蛋白质，它们对空肠梭菌的运动至关重要。为了帮助理解它们的功能并确定它们是否是质周支架的一部分，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构建马达的进化史。乳杆菌弧菌是一种与PflA有潜在远缘同源的亲缘菌。确定该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，以及马达是否来自其他辅助蛋白结合前的早期前体。其次，体外工作将提供更高分辨率的蛋白质结构域和原子结构信息。要成为复合体的一部分，蛋白质必须进化出一个结合界面。为了确定质周鞭毛蛋白之间的蛋白质结合界面（例如PflAB， MotB），我将对被认为相互作用的蛋白质对进行下拉测定，使用不同的截断来确定相互作用区域。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，在硅系统发育和序列分析将补充湿实验室研究。为了建立epsiln - proteobacterian马达的进化史，并帮助确定外周蛋白的募集顺序，我们将建立辅助蛋白的系统发育，并与核心鞭毛组分的系统发育进行比较。PflAB含有许多串联TPR基序，重复序列通常存在于蛋白质结合界面。我们将预测PflAB蛋白结合区域，比较基序之间的序列和dn/ds比率（非同义和同义突变率），这将增强我们对PflAB在马达中的功能机制的理解。这些结构、生物信息学、系统发育和分子生物学实验将对这些蛋白质在鞭毛马达中的作用、它们如何发挥这些功能、它们的进化机制和结合途径提供大量信息，有助于我们的研究

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