High Resolution Cryo-Electron Microscopy of Clathrin Cage Networks

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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.