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Structure and Function of Prokaryotic Appendages by Cryo-Electron Tomography

冷冻电子断层扫描原核附属物的结构和功能

基本信息

批准号：
9745148
负责人：
ELIZABETH R WRIGHT
金额：
$ 8.57万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2020-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9745148
关键词：
Structure Function Prokaryotic Appendages Cryo

项目摘要

PROJECT SUMMARY/ABSTRACT – No change from original proposal 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 variation 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 nonpathogenic 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 the 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.

项目概要/摘要-与原始提案相比无变更许多原核生物利用细胞外的附属物，特别是鞭毛和皮利，用于细菌的运动和定殖。生物和非生物基质。鞭毛和皮利都是许多致病菌的毒力因子细菌种类在这里，我们的目标是确定两件事：1）如何冗余的鞭毛蛋白在多组分鞭毛丝改变了整个鞭毛的结构和功能，以及2）存在的结构变异 IV型皮利的几个类别之间的大分子水平。为了回答这些问题，我们正在使用几种细菌物种，霍乱弧菌和创伤弧菌，引起严重的人类病原体，胃肠炎、腹泻和死亡;以及新月柄杆菌，一种成熟的非致病性模型系统我们正在利用这些生物来建立我们以前对南极洲极地附属物的结构研究。 C. crescentus和发展弧菌物种作为模型系统的病原体的结构研究。我们一起努力，这些研究将为原核生物鞭毛的结构和功能多样性提供新的信息，皮利。该项目将调查的三个领域是： 1.确定致病性和致病性微生物中多鞭毛蛋白鞭毛的结构和功能意义，非致病性细菌实验将确定野生型和突变型C. crescentus、V. cholecystis和V. vulnificus的细胞。冷冻电镜和螺旋重建方法将确定从C. crescentus，V. 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。在这方面的实验目的是确定霍乱弧菌和创伤弧菌T4bP和C. 通过冷冻ET和子断层图像平均来观察新月体。我们将描述结构变化，从突变体到菌毛丝或菌毛复合体及其对菌毛结构、菌毛蛋白分泌和可能的收缩和复杂的整合到细胞膜中。 3.继续开发低温电子断层扫描技术，以改进结构原核生物和它们的附属物。我们的目标是优化我们新安装的Zernike相位对比电子显微镜用于冷冻ET应用。我们的目标是通过优化薄膜结构来发展ZPC低温电子晶体管用于Zernike相位板和开发生物数据收集的全自动化。此外，这个国家这项技术要求开发新的软件和改进现有的算法用于冷冻ET数据的更有效和生物相关的过滤和"去噪"。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy.

DOI：
10.1016/j.jsb.2021.107709
发表时间：
2021-06
期刊：
Journal of structural biology
影响因子：
3
作者：
Yang JE;Larson MR;Sibert BS;Shrum S;Wright ER
通讯作者：
Wright ER

Developing Technologies for Correlative Cryo-Imaging Pipelines.

开发相关冷冻成像管道技术。

DOI：
10.1093/micmic/ozad067.519
发表时间：
2023
期刊：
Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
影响因子：
0
作者：
Wright,ElizabethR;Yang,Jae;Sibert,Bryan;Larson,Matthew;Kim,JosephY;Parrell,Daniel;Sanchez,JuanC;Kumar,Anil;Cai,Kai
通讯作者：
Cai,Kai

Cryo-electron tomography of bacterial viruses.

DOI：
10.1016/j.virol.2012.08.022
发表时间：
2013-01-05
期刊：
Virology
影响因子：
3.7
作者：
Guerrero-Ferreira RC;Wright ER
通讯作者：
Wright ER

The Ms6 Mycolyl-Arabinogalactan Esterase LysB is Essential for an Efficient Mycobacteriophage-Induced Lysis.

DOI：
10.3390/v9110343
发表时间：
2017-11-17
期刊：
Viruses
影响因子：
0
作者：
Gigante AM;Hampton CM;Dillard RS;Gil F;Catalão MJ;Moniz-Pereira J;Wright ER;Pimentel M
通讯作者：
Pimentel M

Morphological characterization of elastin-mimetic block copolymers utilizing cryo- and cryoetch-HRSEM.

利用冷冻和冷冻蚀刻 HRSEM 对弹性蛋白模拟嵌段共聚物进行形态表征。