Structure and function of prokaryotic appendages by cryo-electron tomography

描述（由申请人提供）：许多原核生物使用细胞外附属物，特别是鞭毛和皮利，用于生物和非生物基质的运动和定殖。两 鞭毛和皮利是许多致病细菌物种的众所周知的毒力因子。在这里，我们的目标是确定两件事：1）多组分鞭毛丝中鞭毛蛋白的冗余如何改变整体鞭毛结构和功能，以及2）在大分子水平上存在于几类IV型皮利之间的结构变异。为了回答这些问题，我们正在使用几种细菌，霍乱弧菌和创伤弧菌，人类病原体，导致严重的胃肠炎，腹泻和死亡;和新月柄杆菌，一个完善的非致病性模型系统。我们正在利用这些生物建立在我们以前对C的极地附属物的结构研究基础上。crescentus和发展弧菌物种作为模型系统的病原体的结构研究。总之，这些研究将提供有关原核生物鞭毛和皮利的结构和功能多样性的新信息。本项目将研究的三个方面是：1。确定致病性和非致病性细菌中多鞭毛蛋白鞭毛的结构和功能意义。实验将确定野生型和突变型C. crescentus、V. cholecystis和V. vulnificus的细胞。Cryo-EM和螺旋重建方法将确定从C.新月形静脉、霍乱弧菌和创伤弧菌。2.确定完整细菌细胞中几类IV型皮利的结构。本实验将确定霍乱弧菌和创伤弧菌T4 bP的原位结构和C.通过冷冻ET和子断层图像平均来观察新月体。我们将表征由突变体引起的菌毛丝或菌毛复合体的结构变化及其对菌毛结构、菌毛蛋白分泌和可能的收缩以及复合体整合到细胞膜中的影响。3.继续发展低温电子断层扫描技术，以改进原核生物及其附属物的结构。我们的目标是优化我们新安装的Zernike相衬电子显微镜的冷冻ET应用。我们的目标是通过优化用于Zernike相位板的薄膜和开发生物数据收集的全自动化来开发ZPC cryo-ET。此外，这种最先进的技术要求开发新的软件和改进现有的算法，以实现更有效和生物相关的过滤 和低温ET数据的“去噪”。

DESCRIPTION (provided by applicant): Many prokaryotes use extracellular appendages, notably the flagella and pili, for motility and colonization of biotic and abiotic substrates. Both flagella and pili are well-known virulence factors for numerous pathogenic bacterial species. Here we aim to determine two things: 1) how redundancy of the flagellins in multi-component flagellar filaments alters overall flagellum structure and function, and 2) the structural variatio that exists between several classes of type IV pili at the macromolecular level. To answer these questions, we are using several bacterial species, Vibrio cholerae and Vibrio vulnificus, human pathogens that cause severe gastroenteritis, diarrhea, and death; and Caulobacter crescentus, a well-established non-pathogenic model system. We are using these organisms to build upon our previous structural studies of the polar appendages of C. crescentus and to develop Vibrio species as model systems for structural studies of pathogens. Together, these studies will provide new information regarding the structural and functional diversity of prokaryotic flagella and pili. The three areas to be investigated in this project are: 1. Determine the structural and functional implications of multi-flagellin flagella in pathogenic and non-pathogenic bacteria. Experiments will determine the structure of flagella from wild type and mutant C. crescentus, V. cholerae, and V. vulnificus in situ through cryo-ET of frozen hydrated cells. Cryo-EM and helical reconstruction approaches will define the high-resolution structure of flagella isolated from C. crescentus, V. cholerae, and V. vulnificus. 2. Determine the structures of several classes of type IV pili in intact bacterial cells. Experiments in this aim will determine the in situ structure of he T4bP of V. cholerae and V. vulnificus and Flp-type pili of C. crescentus through cryo-ET and sub-tomogram averaging. We will characterize structural changes resulting from mutants to the pilus filament or pilus complex and their impact on pilus structure, pilin secretion and possible retraction, and complex integration into the cell membrane. 3. Continue the development of cryo-electron tomography technology to generate improved structures of prokaryotes and their appendages. We aim to optimize our newly installed Zernike phase contrast electron microscope for cryo-ET applications. Our goal is to develop ZPC cryo-ET by optimizing the thin films used for Zernike phase plates and developing full automation of biological data collection. In addition, this state of the art technology calls for the development of new software and improvement of already existing algorithms for a more efficient and biologically relevant filtering and 'denoising' of cryo-ET data.