Toward Atomic Resolution of Membranes and Membrane-Associated Machines

膜和膜相关机器的原子分辨率

基本信息

批准号：
9117230
负责人：
Adam Frost
金额：
$ 170.65万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
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：
2013-09-30 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9117230
关键词：
Toward Atomic Resolution Membranes Membrane

项目摘要

DESCRIPTION (provided by applicant): The unifying goal of this proposal is to understand the structures and functions of machines that assemble on cellular membranes. Cells depend upon these machines in order to transform the shape, size and connectivity of their membranes and such "remodeling" underlies cell division, migration, differentiation, and communication. In addition, every pathogen hijacks or disrupts membrane-associated complexes to infect or escape from cells. Despite such central importance we lack comprehensive models of how membrane-binding proteins transduce signals, oligomerize, or remodel the size, shape and topology of cellular membranes. To overcome the intrinsic challenges in studying multi-component complexes that assemble transiently on membranes, we will develop genetic, biochemical and structural methods to discover and characterize bilayer-bound machines in molecular detail and to learn how they function within intricate cellular pathways. We build genome-scale maps of genetic interaction networks in model organisms to identify multi-component complexes and to infer their functions. We develop methods for reconstituting membrane-binding proteins in the presence of model membranes that mimic the in vivo target membrane in topology, lipid composition, size, and shape. We then leverage advances in cryo-electron microscopy (cryoEM) with our novel image analysis algorithms to solve structures of these machines in their native, membrane-associated states. These innovative tools are distinct methodologically but reinforcing: our genetic maps first define complexes for in depth in vitro study; and after reconstitute and solve 3D reconstructions by cryoEM we probe the functional predictions of our models with loss-of-function versus second-site suppression assays in vivo. These orthogonal approaches thus provide synergistic evidence for the mechanisms that drive cellular functions and result in genuine atomic-resolution understanding of the structures and functions we study. In concert with technology advances in electron optics and electron detectors, my laboratory is overcoming the genetic, biochemical and computational challenges responsible for the lack of structural models for membrane-bound protein assemblies. The aims outlined here are first, critical steps in our long-term research program to determine how the mechanisms we discover are corrupted by disease or hijacked by pathogens. Our ultimate objective is to translate this understanding into effective and tolerable treatments for diseases caused by defective or co-opted membrane-associated machines.

描述（由申请人提供）：该提案的统一目标是了解聚集在细胞膜上的机器的结构和功能。细胞取决于这些机器，以改变其膜的形状，大小和连通性以及这种“重塑”是细胞分裂，迁移，分化和通信的基础。另外，每种病原体劫持或破坏膜相关的复合物，以感染或逃脱细胞。尽管至关重要，但我们缺乏膜结合蛋白如何转导信号，寡聚或重塑细胞膜的大小，形状和拓扑的全面模型。为了克服在研究膜上瞬时组装的多组分复合物中的内在挑战，我们将开发遗传，生化和结构方法，以分子细节发现和表征双层结合的机器，并学习它们在复杂的细胞途径中的功能。我们在模型生物体中构建了遗传相互作用网络的基因组规模图，以识别多组分复合物并推断其功能。我们开发了在存在模型膜的情况下重建膜结合蛋白的方法，这些膜模仿了拓扑，脂质组成，大小和形状中的体内靶膜。然后，我们利用新型的图像分析算法利用冷冻电子显微镜（Cryoem）的进步来求解这些机器与膜相关状态的这些机器的结构。这些创新的工具在方法论上是独特的，但可以加强：我们的遗传图首先定义了体外研究深度的复合物；在Cryoem重新构建和解决3D重建之后，我们探测了模型的功能预测，其功能丧失与第二站点抑制分析在体内。因此，这些正交方法为驱动细胞功能的机制提供了协同证据，并导致对我们研究的结构和功能的真正原子分辨率的理解。在电子光学和电子探测器方面的技术进步时，我的实验室正在克服导致缺乏膜结合蛋白质组件结构模型的遗传，生化和计算挑战。这里概述的目的是我们长期研究计划中的关键步骤，以确定我们发现的机制如何被疾病腐败或被病原体劫持。我们的最终目标是将这种理解转化为由缺陷或共同膜相关的机器引起的有效且可忍受的治疗方法。