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HIV-1 assembles at and buds through the cell membrane. The released particle is non-infectious until the viral protease cleaves the main structural protein, Gag, in five places. This causes a structural rearrangement in the virus called “maturation”, to form the infectious virion. The viral particles are variable in size and shape. We are studying how HIV-1 assembles and matures by using cryo-electron tomography to reveal the internal structure of the virus. For examples see Schur et al. 2016, Mattei et al. 2016.
Influenza virus particles can be long filaments or small round structures depending on the properties of the virus and of the infected cells. We are studying the roles of the different viral proteins in assembling virus particles and determining their shape. We are trying to determine the structures of the viral proteins directly within virus particles. For example see Peukes et al. 2020.
Coated membrane vesicles transport material between the different compartments of a cell. We study COPI-coated vesicles and clathrin-coated vesicles. We have reconstituted the budding of coated vesicles in vitro and are using these systems to understand the coat structure. We are also applying cryo-electron tomography to cells embedded in resin or thinned using focussed-ion-beam milling, allowing us to obtain structures for coated vesicles within the cell. For examples see Avinoam et al. 2015, Dodonova et al. 2017, Bykov et al. 2017, Kovtun et al. 2018, Kovtun et al. 2020.
We have a project funded by the European Research Council to study the mechanisms of membrane fusion. This work encompasses studies on regulated SNARE-mediated fusion at neuronal synapses, as well as mechanisms of fusion of enveloped viruses with target cell membranes. We apply cryo-electron tomography to study viral fusion proteins, as well as to visualize fusion reconstituted in vitro using purified proteins and lipids. For example see Bharat et al. 2014.
We are attempting to optimize and improve the way in which cryo-electron tomography data is collected at the microscope, and how the data is processed using subtomogram averaging methods. Through methods development projects we have been able to obtain higher resolution structures from smaller amounts of data, both in vitro and within the cell. For examples see Hagen et al. 2017, Turonova et al. 2017.
We have also developed methods for correlative fluorescence and electron microscopy, and for cryo-fluorescence microscopy. For examples see Kukulski et al. 2012, Schorb et al. 2017, Bykov et al. 2019.