Research summary

We are interested in studying the three-dimensional structures of large macromolecular machineries: their structural architectures, the regulation of their function and the dynamic processes of their assembly and disassembly, by molecular electron microscopy. A full understanding of the biological functions/processes of macromolecular machinery requires structural information at a wide range of resolutions, from the atomic details of all of its components, the spatial arrangements of these components and the interactions between them, all the way to its cellular context. X-ray crystallography and NMR spectroscopy are the traditional methods used to determine the structures of macromolecules at atomic resolution. Molecular electron cryomicroscopy (cryoEM) can be used to study the macromolecular machineries in their native conformations at a wide resolution range, from sub-nanometer resolution (single particle cryoEM) to the cellular level (electron tomography). Thus the cryoEM has the potential to integrate the structural information obtained at different resolution levels.

Of particular interest for us are the endocytotic machinery (clathrin-coated vesicles) and the protein degradation machinery (proteasome):

Endocytotic machinery

Clathrin-coated vesicles are endocytotic machineries found in all eukaryotic cells, from yeast to human. In clathrin-mediated endocytosis, clathrin-coated vesicles transport lipid and selected cargo molecules from the plasma membrane to endosomes. Animal cells use this pathway to uptake extracellular molecules such as low-density lipoprotein that transports cholesterol. Many viruses, e.g. influenza, are known to infect host cells via this endocytotic pathway as well. Clathrin-coated vesicles also carry the secretory traffic from the trans-Golgi network to endosomes. In the synapses of nervous system they participate in the recycling of synaptic vesicles. We are using cryoEM to dissect the complex and dynamic process of clathrin-mediated endocytosis, by capturing the snapshots of clathrin-coated vesicles in the dynamic process of assembly (coated vesicle formation) and disassembly (un-coating).

Protein degradation machinery

In all eukaryotic cells, 26S proteasome catalyzes most intracellular protein degradation in an ATP dependent manner. The 26S proteasome is composed of a 20S protease core particle (CP) sandwiched between two 19S regulatory complexes (RP). The atomic structure of 20S CP has been determined by the x-ray crystallography, and the low-resolution shape of the 19S RP has been determined by the cryoEM. However, the structures/functions of many subunits in the 19S RP remain to be elucidated at a higher resolution level. We are interested in studying structure/function of the 19S RP. Among the subunits in the 19S, the proteasomal ATPases recognize the substrates targeted for the degradations, unfold globular substrates, induce gate-opening in the 20S, and facilitate translocation of the unfolded substrate into 20S CP for degradation. We are using single particle cryoEM together with other biochemical methods to study the structure/function of the proteasomal ATPases and its mechanism of inducing the gate-opening in the 20S core particle.