References:

Liu J, Lin T, Botkin DJ, McCrum E, Winkler H, Norris SJ: Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C Ring Assembly Flexion, J Bacteriol 2009 191(16):5026-36.
Wu S, Liu J, Reedy MC, Winkler H, Reedy MK, Taylor KA: Methods for Identifying and Averaging Variable Molecular Conformations in Tomograms of Actively Contracting Insect Flight Muscle. J Struct Biol 2009 Aug 19. [Epub ahead of print].
Sanabria H, Swulius MT, Kolodziej SJ, Liu J, Waxham NM: CaMKII Regulates Actin Assembly and Structure, J Biol Chem 2009 284(15):9770-80.
Winkler H, Zhu P, Liu J, Ye F, Roux KH, Taylor KA: Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes. J Struct Biol 2009 165(2):64-77.
Liu J, Bartesaghi A, Borgnia M, Sapiro G, Subramaniam S: Molecular architecture of native HIV-1 gp120 trimers. Nature 2008 455:109-113.
Hampton CM, Liu J, Taylor DW, DeRosier DJ, Taylor KA: The 3D Structure of Villin as a Unique F-Actin Crosslinker. Structure. 2008 16(12):1882-91.
Ye F, Liu J, Winkler H, Taylor KA: Integrin IIba3 in a membrane environment remains the same height after Mn2+ Activation when observed by cryo-electron tomography. J Mol Biol 2008 378:976-86.
Miao L, Vanderlinde O, Liu J, Grant R, Wouterse A, Philipse A, Stewart M, Roberts TM: Filament packing constraints generate protrusive force in amoeboid cell motility. PNAS 2008 105:5390-5.
Bartesaghi A, Sprechmann P, Liu J, Randall G, Sapiro G, Subramaniam S: Classification and 3D averaging with missing wedge-correction in biological electron tomography. J Struct Biol 2008162:436-50
Dai W, Jia Q, Bortz E, Shah S, Liu J, Atanasov I, Li X, Taylor KA, Sun R, Zhou ZH: Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy. J Struct Biol 2008 161:428-438.
Liu J, McBride MJ, Subramaniam S: Cell-surface filaments of the gliding bacterium Flavobacterium johnsoniae revealed by cryo-electron tomography. J Bacteriol 2007 189: 7503-7506.
Subramaniam S, Bartesaghi A, Liu J, Bennett AE, and Sougrat R: Electron tomography of viruses. Current Opinion in Structural Biology 2007 5:596-602.
Bennett AE, Liu J, Van Ryk D, Bliss D, Arthos J, Henderson R M, Subramaniam S: Cryo electron tomographic analysis of an HIV neutralizing protein and its complex with native viral gp120. J Biol Chem 2007, 282(38): 27754-27759.
Liu J, Taylor DW, Krementsova EB, Trybus KM, Taylor KA: Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography. Nature 2006, 442:208-211.
Zhu P, Liu J, Bess J, Jr., Chertova E, Lifson JD, Grise H, Ofek GA, Taylor KA, Roux KH: Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 2006, 441:847-852
Liu J, Wu S, Reedy MC, Winkler H, Lucaveche C, Cheng Y, Reedy MK, Taylor KA: Electron tomography of swollen rigor fibers of insect flight muscle reveals a short and variably angled S2 domain. J Mol Biol 2006, 362:844-860
Liu J, Reedy MC, Goldman YE, Franzini-Armstrong C, Sasaki H, Tregear RT, Lucaveche C, Winkler H, Baumann BA, Squire JM, Irving TC, Reedy MK, Taylor KA: Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. J Struct Biol 2004, 147:268-282
Liu J, Taylor DW, Taylor KA: A 3-D reconstruction of smooth muscle alpha-actinin by CryoEm reveals two different conformations at the actin-binding region. J Mol Biol 2004, 338:115-125
Tama F, Feig M, Liu J, Brooks CL, 3rd, Taylor KA: The requirement for mechanical coupling between head and S2 domains in smooth muscle myosin ATPase regulation and its implications for dimeric motor function. J Mol Biol 2005, 345:837-854
Liu J, Wendt T, Taylor D, Taylor K: Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction. J Mol Biol 2003, 329:963-972
21. Zhu P, Chertova E, Bess J, Jr., Lifson JD, Arthur LO, Liu J, Taylor KA, Roux KH: Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions. PNAS 2003, 100:15812-15817
Book chapter:
Goldstein SF, Li C, Liu J, Miller M, Motaleb M, Norris SJ, Silversmith RE, Wolgemuth CW, Charon NW: The Chic Motility and Chemotaxis of Borrelia burgdorferi. In: Borrelia: Molecular and Cellular Biology, Edited by D. Scott Samuels, in press.

Jun Liu, PhD

Assistant Professor
Pathology & Laboratory Medicine
(713) 500 - 5342
Jun.Liu.1@uth.tmc.edu

3-D structure/function of nano-machines in situ
AIDS virus
Viral entry and antibody neutralization
Lyme-disease spirochete
Flagellar motor

Education: 1998, Chinese Academy of Sciences, Beijing, China

Research Interests: Infectious diseases continue to adversely affect human health, and despite significant advances in medical science, they remain the leading cause of death. Our long-term goal is to open the avenue to the understanding the molecular basis of infectious diseases by studying the three-dimensional structural organization of infectious pathogens (including bacteria and viruses).

High-throughput Cryo-Electron Tomography

Cryo-Electron Tomography (cryo-ET) is currently the most promising technology to determine 3-D architecture of nano-machines in situ at molecular resolution. We have established and optimized a high-throughput Cryo-ET system (Figure 1), which has been used effectively to collect cryo tomograms of intact B. burgdorferi cells embedded in thin vitreous ice. We have developed the unique procedure to visualize hundreds of cells at molecular resolution within one week, using a combination of automated Cryo-ET data collection and high-throughput image processing. The ability to rapidly produce and process hundreds of tilt series has improved both the throughput and the quality of the resulting 3-D reconstructions. It’s becoming possible to generate a high-resolution 3-D structure of molecular nano-machine in situ within a month.

High-throughput pipeline of Cryo-ET

Figure 1. High-throughput pipeline of Cryo-ET: from sample to high resolution 3-D structure in situ. Preparation of frozen hydrated specimen of viable B. burgdorferi cells (A) and the loading of multiple EM grids into a TEM microscope (B) takes about one hour. Nearly 100 cryo tomography tilt series of intact cells (C) can be generated by FEI batch tomography in three days. In three weeks, we are able to process the resulting data and determine the molecular architecture of the intact flagellar motor in situ (D).

Lyme Disease Spirochete: Borrelia burgdorferi

B. burgdorferi belongs to a group of bacteria, called spirochetes, which are medically significant, but poorly understood. These organisms cause several major diseases in humans: syphilis (T. pallidum), Lyme disease (B. burgdorferi), and leptospirosis (L. interrogans). Syphilis is a major health problem in the world. Leptospirosis is the most common worldwide waterborne zoonosis. Lyme disease is the most common vector-borne infection in the United States, and has shown a steady increase in incidence since its discovery in 1975. Motility is an essential component of the pathogenesis of these and other bacteria and the flagellar motor is considered to be a proficient biological machine for this purpose.

We have focused on B. burgdorferi and its periplasmic flagella as a model system to study the flagellar structure and motility in situ. In collaboration with Dr. Steven Norris, we were able to visualize the three-dimensional structures of intact B. burgdorferi cells with unprecedented detail (as shown in Fig. 2), by using high-throughput cryo-ET.

Video: Surface views of flagellar motor are colored in light blue (stator and C ring), and beige (export apparatus)

By averaging the 3-D images of ~1280 flagellar motors, a ~3.5 nm resolution model of the stator and rotor structures was obtained (see video above). The results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C ring assembly during flagellar rotation. Transposon mutants of flgI lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Therefore, combining genetics with our advanced imaging methods will permit a thorough analysis of the structure and assembly of the flagellar motor in situ at the molecular level.

Structural basis of HIV viral entry and antibody neutralization

Interaction between HIV/SIV and broadly neutralizing antibodies revealed by cryo-ET

The envelope glycoproteins (Envs) of HIV/SIV mediate binding to CD4 and co-receptors on target cells to initiate viral entry and infection. The Envs comprise the functional trimeric spikes on the surface of the viral membrane and is composed of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). Although several crystal structures of gp120 monomer are known, the crystal structure of the HIV-1 Env trimer has not yet been determined. Our main goal is to provide molecular understanding of the mechanisms of HIV-1 antibody neutralization at its most fundamental level – the three-dimensional structures of native Envs and their interaction with broadly neutralizing antibodies, by using an approach outlined in Fig.3. A deeper understanding of how broad neutralization is accomplished could provide significant insights on the development of mimics of the viral envelope spike and the rational design of novel immunogen specifically tailored to elicit broadly neutralizing antibody responses against HIV.

Figure 4 CD4 binding results in a major opening of Env trimer of HIV revealed by Cryo-ET. These changes promote viral entry into host cell. [Liu et al. 2008 Nature]
CD4 binding results in a major opening of Env trimer of HIV revealed by Cryo-ET. These changes promote viral entry into host cell. [Liu et al. 2008 Nature]

Recently we determined the molecular architecture of native HIV-1 gp120 trimers on the envelope of virions [Liu et al. 2008 Nature], by using high-throughput cryo-ET. For the first time, we were able to demonstrate the structural and conformational changes that occur upon the binding of the Env spikes of HIV to its host cell surface receptor (CD4) (Fig.4) at 2.0nm resolution. The binding of Env to CD4 results in a major reorganization of the Env trimer and a close contact between the virus and target cell co-receptor, thus facilitating viral entry.
In addition, the novel techniques utilized in the projects will foster an in-depth understanding of a variety of human pathogens and offer a wide spectrum of important biomedical information at molecular resolution in living organisms.