In the very crowded inner environment of a cell, most macromolecules function in the form of complexes, many being described as “molecular machines.” To understand the machines’ structures and structural changes that occur during the working cycle, we employ cryo-electron microscopy to visualize them as “single particles” or ordered functional assemblies. The micrographs are analyzed by computational image processing to reveal the structures and conformational variations of these molecules. We then combine the structural information with data from accompanying biophysical and biochemical techniques to elucidate the mechanisms of these large macromolecular machines.

Our current research focuses on three topics:

A. The methodology development for more efficient and high resolution cryo-electron microscopy. As a group using cryo-EM as the major tool, we are devoted to new method implementation and application depending on the nature of different samples that we work on. We are aiming at pushing the boundary of the technology towards solving high resolution structures of macromolecules with small molecular weight and flexibility.

1. Exploiting near-atomic resolution single particle reconstruction of macromolecules with small size.

2.Developing novel supporting grids with bioactive functionalized graphene monolayer for high-resolution cryo-EM structural determination.

3.Developing novel imaging methods to obtain atomic-resolution structures of macromolecules.

4. Implementing new algorithms dealing with conformational heterogeneity in single particle cryo-EM analysis.

B. The mechanism and regulations of nucleic acid quality control. We have been using electron microscopy as our major tool to study the structure and mechanism of macromolecular complexes involved in RNA metabolism, including the human RISC-loading complex (RLC) for small RNA biogenesis in RNA interference pathway, the eukaryotic exosome complex responsible for RNA degradation, the Ro-Y-RNA-PNPase complex (RYPER) in RNA quality control, and more recently, the Group II Intron RNP complex.

1.The high-resolution structures and mechanism of human Dicer and its complex with RNA substrates.

2. Architecture and mechanism of the RISC-loading complex (RLC) in RNA interference pathways.

3. Mechanisms and regulations of exosome-mediated RNA processing and degradation.

4. Structure and mechanism of Group II Intron RNP complex.

5.Structure and mechanism of human RAD51 in DNA homologous recombination.

C. The coordination mechanisms of cytoskeleton and membrane systems. The complex cytoskeleton systems including microtubules, actin networks and cell membranes work together in many vital physiological processes. The coordination among these systems is highly regulated and essential in cell shape and polarity definition, cell migration, cell division, and intra-cellular vesicle trafficking and so on. We study the coordination mechanisms among microtubules, actin networks, and cell membranes with cryo-EM.

1.The near atomic Cryo-EM structure and mechanism of tethering complex exocyst.

2.The mechanism of WHAMM in coordinating cytoskeleton systems in membrane deformation.

3.WHAMM initiates autolysosome tubulation by promoting actin polymerization on autolysosomes.