Structure and function of prokaryotic appendages by cryo-electron tomography

描述(申请人提供)：许多原核生物使用胞外附属物，特别是鞭毛和菌毛，来移动和定植生物和非生物底物。两者都有 鞭毛和菌毛是许多致病细菌的已知毒力因子。在这里，我们旨在确定两件事：1)多组分鞭毛细丝中鞭毛蛋白的冗余如何改变整个鞭毛的结构和功能；2)在大分子水平上存在于几类IV型鞭毛之间的结构差异。为了回答这些问题，我们使用了几种细菌，霍乱弧菌和创伤弧菌，这是人类导致严重胃肠炎、腹泻和死亡的病原体；以及新月弯杆菌，一个成熟的非致病模型系统。我们正在利用这些生物来建立我们以前对新月浑圆线虫极地附肢的结构研究，并开发弧菌物种作为病原体结构研究的模式系统。总之，这些研究将为原核生物鞭毛和菌毛的结构和功能多样性提供新的信息。本项目拟研究的三个方面是：1.确定多鞭毛在致病和非致病细菌中的结构和功能意义。实验将通过冷冻水化细胞原位测定野生型和突变型新月浑圆线虫、霍乱弧菌和创伤弧菌的鞭毛结构。冷冻-EM和螺旋重建方法将定义从新月形弧菌、霍乱弧菌和创伤弧菌分离的鞭毛的高分辨率结构。2.确定了几类IV型菌毛在完整细菌细胞中的结构。本实验将通过冷冻法和亚层析平均法确定霍乱弧菌和创伤弧菌的T4bP和新月形弧菌的FLP型菌毛的原位结构。我们将描述由菌毛细丝或菌毛复合体突变引起的结构变化，以及它们对菌毛结构、菌毛分泌和可能的回缩以及复合体整合到细胞膜中的影响。3.继续发展冷冻电子断层扫描技术，以产生原核生物及其附件的改进结构。我们的目标是优化我们新安装的Zernike相差电子显微镜，用于低温应用。我们的目标是通过优化用于Zernike相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，以实现更有效和与生物相关的过滤 以及对低温数据进行去噪处理。

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.