Cryo-EM technology development
In recent years, the most exciting technological breakthroughs in single particle cryo-EM were brought about by the broad application of direct electron detection cameras. All current commercial direct electron detection cameras have superior detective quantum efficiency (DQE) at all frequencies over traditional scintillator based digital cameras 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's laboratory at UCSF, we developed the programs MotionCorr (Li et al. 2013, Nature Methods) and MotionCor2 (Zheng et al. 2017) for fast and accurate correction of beam-induced image motion.
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Membrane protein structure and function
We are interested in developing novel technologies to enable high-resolution structure determination of integral membrane proteins, particularly in lipid bilayer environments. Together with Charles Craik's 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's laboratory at UCSF, we demonstrated that lipid-nanodiscs can be used for high-resolution structure determination of membrane proteins and visualization of 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 environments, such as using Saposin as a scaffolding protein to reconstitute lipid-protein complexes for single particle cryo-EM structure determination (Frauenfeld et al., 2016, Nature Methods). (Add native nanodiscs)
Together with David Julius’s laboratory at UCSF, we are studying structures of various members of the transient receptor potential (TRP) channel superfamily. We have now determined atomic structures of TRPV1 (Liao, et al. 2016, Nature; Cao et al. 2013 Nature and Gao et al. 2016, Nature), TRPV5 (Dang et al. 2019, PNAS), TRPA1 (Paulsen et al. 2015, Nature, Zhao et al. 2020, Nature), TRPM4 (Autzen et al. 2018, Science), and TRPM8 (Diver et al. 2019, Science). These structures have shed light on how different TRP channels are activated and inhibited by small molecules, toxins, and small proteins such as calmodulin. Shared mechanisms of modulation by calcium ions were also revealed in the TRPM structures.
We are also studying structures of ABC transporters (Kim et al. 2015, Nature). Together with a number of other laboratories at UCSF, including the laboratories of Charles Craik and Robert Stroud, we formed a consortium with the goal to study structures and functions of various ABC transporters.
Jan lab collaborations
Collaborations and shared students with Aashish Manglik’s lab have begun investigations into G-protein coupled receptor (GPCR) complexes and transporters. Specifically, structures of the only mammalian ion exporter, ferroportin, are being investigated to learn more about the inhibitory mechanism of its endogenous antagonist, hepcidin, and other small molecule inhibitors. GPCR structure determination has seen a revolution over the past few years driven my cryo-EM. We are now leveraging new biochemical stabilization techniques and advancing EM technology to determine structures that can be used to better understand GPCRs with unique extracellular domains.
X lab collaboration
Nucleosome remodelers
Narlikar lab collaboration
Protein degradation machinery
In all eukaryotic cells, the 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 the 20S CP has been determined by the x-ray crystallography, and the low-resolution shape of the 19S RP has been determined by the cryo-EM. However, the structures/functions of many subunits in the 19S RP remain to be elucidated at higher resolution. 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 degradation, unfold globular substrates, induce gate-opening in the 20S, and facilitate translocation of the unfolded substrate into the 20S CP for degradation. We are using single particle cryo-EM together with other biochemical methods to study the structure/function of the proteasomal ATPases and their mechanisms of inducing gate-opening in the 20S CP.