The Carter Group
Unravelling the structure of the viral proteins mediating replication and infection in the host-cell hold the key to understanding the fundamental biological properties of viruses. Our work focusses on better understanding virus-host interactions by visualising them in a frozen-hydrated state at macromolecular resolution using cryo-electron tomography (cryo-ET).
The aim of our work is to establish cutting-edge in situ imaging technologies, such as cryo-CLEM and cryo-FIB milling to image the replication cycles of clinically important envelope viruses inside the cell in three-dimensions and at high-resolution. In the simplest cryo-CLEM workflow, mammalian cells expressing an engineered fluorescent virally encoded protein are plunge-frozen and imaged in a cryo-fluorescence microscope to identity the location of bright puncta in the thin-edges of cells. The frozen sample is then transferred into the cryo-electron microscope and the target is uncovered by correlating the light and electron microscopy images and imaged finally at high resolution in 3D by cryo-ET. To uncover a target buried deep in a cell, there are ways to integrate cryo-CLEM and cryo-FIB milling. These workflows allow the use fluorescence-guided FIB milling to reliably capture a fluorescently-labeled structure within a 100-200 nm-thick lamella for subsequent cryo-ET imaging.