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High Resolution Cryo-Electron Microscopy of Clathrin Cage Networks

网格蛋白笼网络的高分辨率冷冻电子显微镜

基本信息

批准号：
1786909
负责人：
金额：
--
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1786909
关键词：
Resolution Cryo Electron Microscopy Clathrin

项目摘要

In cryo-electron microscopy, the superior signal sensitivity of new direct electron detectors has revolutionised the field of structure determination allowing sub-4Å structures of challenging targets such as membrane proteins and ribosomes to be obtained without using X-ray crystallography. Clathrin-mediated endocytosis is a fascinating mechanical phenomenon that drives the selective internalisation of molecules into cells. In order to work properly, clathrin-mediated endocytosis requires accurate and timely assembly of a clathrin lattice and coordination with a network of more than 20 adaptor proteins to form a coated vesicle which will be able to select molecules from the outside of the cell for delivery to specific destinations.The aim of this project is to elucidate how the proteins involved in the network of clathrin and its adaptor proteins interact to achieve clathrin-coated vesicle formation by exploiting cryo-electron microscopies capability to carry out high resolution structural analysis of clathrin cage complexes. High resolution 3D cryo-electron microscopy will be used to visualise adaptor proteins binding to clathrin cages as well as biophysical approaches such as dynamic light scattering, time-resolved fluorescence anisotropy and isothermal titrating calorimetry in order to investigate how clathrin-adaptor interactions result in formation of a functional coated vesicle network.

在低温电子显微镜中，新的直接电子探测器的上级信号灵敏度已经彻底改变了结构测定领域，允许在不使用X射线晶体学的情况下获得具有挑战性的目标（如膜蛋白和核糖体）的亚40 μ m结构。网格蛋白介导的内吞作用是一种迷人的机械现象，它驱动分子选择性内化到细胞中。为了正常工作，网格蛋白-介导的内吞作用需要网格蛋白晶格的准确和及时的组装，并与20多个接头蛋白的网络相协调，以形成一个包被的囊泡，该囊泡将能够从细胞外选择分子以递送到特定目的地。本项目的目的是阐明网格蛋白网络中的蛋白质及其接头蛋白如何相互作用，通过利用低温电子显微镜对网格蛋白笼状复合物进行高分辨率结构分析的能力来实现网格蛋白包被的囊泡的形成。高分辨率3D冷冻电子显微镜将用于可视化衔接蛋白结合网格蛋白笼以及生物物理方法，如动态光散射，时间分辨荧光各向异性和等温滴定量热法，以研究网格蛋白衔接相互作用如何导致形成功能性包被囊泡网络。