As a laboratory specializes in electron cryo-microscopy (cryo-EM), we are interested in both methodologies and applications of this versatile technology to study structures and functions of various biological macromolecules. Of particular interests to us are studying integral membrane proteins and large dynamic macromolecular machineries, and developing enabling technologies to facilitate structural studies of these challenging macromolecules by cryo-EM.
In recent years, the most exciting technological breakthrough in single particle cryo-EM were brought by the broad application of direct electron detection camera. All current commercial direct electron detection cameras have superior detective quantum efficiency (DQE) at all frequencies over traditional scintillator based digital camera and photograph films. They also have a high output frame rate, typically between 10 to 40 frames per second. Nowadays cryo-EM images of frozen hydrated biological samples are typically recorded as dose-fractionated movie stacks, which enable correction of beam-induced image motion. Together with David Agard laboratory at UCSF, we developed programs MotionCorr (Li et al. 2013, Nature Methods) and MotionCor2 (Zheng et al. 2017) for fast and accurate correction of beam-induced image motion.
We are interested in developing novel technologies to enable high-resolution structure determination of integral membrane proteins, particularly in lipid bi-layer environment. Together with Charles Craik laboratory at UCSF, we developed a general approach of using conformational specific monoclonal Fabs to facilitate structural studies of small soluble and integral membrane proteins by single particle cryo-EM (Wu et al. 2012, Structure and Kim et al. 2015, Nature). Together with David Julius laboratory at UCSF, we demonstrated that lipid-nanodisc can be used for high-resolution structure determination of membrane proteins, and visualized specific lipid protein interactions (Gao et al. 2016, Nature). We are also investigating other methodologies that enable high-resolution structure determination of integral membrane proteins in lipid environment, such as using Saposin as scaffolding protein to reconstitute lipid-protein complex for single particle cryo-EM structure determinations (Frauenfeld et al., 2016, Nature Methods).
Together with David Julius laboratory at UCSF, we are studying structures of various members of transient receptor potential (TRP) channel superfamily. We have now determined atomic structure of TRPV1 (Liao, et al. 2016, Nature; Cao et al. 2013 Nature and Gao et al. 2016, Nature) and TRPA1 (Paulsen et al. 2015, Nature).
We are also studying structures of ABC transporters (Kim et al. 2015, Nature). Together with a number of other laboratories at UCSF, including Charles Craik and Robert Stroud laboratories, we formed a consortium with the goal to study structures and functions of various ABC transporters.
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.