Cryo-electron tomography of viruses, vesicles and cells.
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Project 1: Cryo-electron tomography of virus or vesicle assembly.
Enveloped viruses and coated membrane trafficking vesicles are extraordinary self-assembling machines. The protein building blocks must interact with one another to collect all relevant components and/or cargo together at the assembly site, they must reshape a lipid bilayer to form the bounding membrane of the virus/vesicle, and they must drive membrane scission to release the virus/vesicle from the donor membrane. The virus/vesicle typically then undergoes a reassembly or disassembly process to prepare for fusion with a target membrane. We wish to obtain a mechanistic understanding of these processes in systems including HIV-1, influenza virus, clathrin-coated vesicles and COPI-coated vesicles.
To do this we develop and apply new approaches for cryo-electron tomography, correlative light and electron microscopy and image processing. These methods allow us to obtain detailed 3D structural information at different stages in assembly under close-to-native conditions, including inside cells.
This project will address either HIV-1 or membrane trafficking vesicles. The PhD student will study the assembly process and the subsequent structural changes required for function. The methods used will be cryo-electron tomography and image processing, as well as other complementary techniques. Depending on the experience and interests of the student, the focus of the project could be directed towards the biological, technical, or computational aspects.
Project 2: Studying membrane fusion mechanisms using cryoEM.
The ability to carry out fusion between two membrane compartments in a cell is fundamental to life. It is also a key step in the infection process for enveloped viruses. We want to understand basic mechanisms of membrane fusion in model systems. In particular we aim to reveal how the fusion machinery is arranged on the membrane, how it changes in structure and arrangement during the fusion process, and how these changes reshape the membrane to drive fusion.
The PhD student will primarily apply cryo-electron tomography to study synaptic fusion machinery reconstituted in vitro. Complementary methods including fluorescence microscopy, correlative light and electron microscopy, and biochemistry will be applied as appropriate. The aim of the project is to generate 3D structural data on multiple intermediate stages in fusion in order to understand how different components of the fusion machinery act in concert to drive the process. Comparative studies may also be performed on influenza virus fusion.
Depending on the experience and interests of the student, there may be some flexibility to focus the project towards biological, biophysical, technical, or computational aspects of the research project.