3D-Electron Microscopy

The determination of cellular organization and protein structures is most significant in both understanding cellular function and developing targeted therapies, an approach termed molecular medicine.  Cryo-electron microscopy (cryoEM) is currently leading a revolution in structural biology providing nanometer and often near atomic resolution of cellular machines and macromolecular complexes.  The Department of Pathology and Laboratory Medicine is equipped with a state-of-the-art cryo-EM facility including a 300kV cryo-electron microscope (Technai F30 Polara) and a new generation direct electron detector (GATAN K2 Summit). Funded by the National Institutes of Health, a high-throughput imaging process pipeline has been developed to allow remote data collection and structural determination of macromolecular complexes at high resolution by cryoEM. Our system is particularly powered by high-speed computing resources, which facilitate offsite data processing ideal for residents interested in computational biology.  This arrangement is well tailored to accommodate the demanding clinical requirements of residents in their efforts to establish a significant research component during their clinical training. Interested medical residents, postdocs, and graduate students should contact Drs. Jun Liu, Xiangan Liu or Dr. William Glass.

The structure-function relationships of bacterial pathogenesis.

Understanding the pathogenesis of Shigella, a leading cause of dysentery worldwide, is crucial to combat developing antibiotic resistance.  Cryo-ET has been employed to resolve the 3D structure of the Shigella injectisome within Shigella minicells (Hu, Morado et al. 2015).

Molecular Basis of viral infection and genome translocation.

Cryo-ET studies are now increasing our understanding of bacteriophage structure, cellular adsorption and genome ejection, aspects that are fundamental to phage life cycle and are crucial steps in the development of bacteriophage treatments for bacterial infections, which are increasingly antibiotic resistance. As shown below, Cryo-ET has been employed to capture T4 virions at successive stages of infection.

3DEM of macromolecules of biological interest.

Atomic resolution X-ray structures of the component enzymes are docked in the EM map of the pyruvate dehydrogenase multienzyme complex consequently rendering high resolution insight into the functional organization of the largest enzyme complex known (10 megadaltons) (24). PDC is the crucial enzyme linking glycolysis and the tricarboxylic acid cycle.  The core functional unit of the enzyme is shown in B) in which the x-ray structure has been docked into the EM map.  The E1 tetramers are shown in yellow and the E2 trimer is in green.  Structural determination of the PDC complex has provided a better understanding of disorders like PDC deficiency and may lead to development of targeted therapies in the future.

Webpage organized by Madeline M Farley, Ph.D. and James K Stoops, Ph.D. with special thanks to Jun Liu, Ph.D. and William Glass, M.D, Ph.D., MBA.

Selected Publications

  1. Liu, J., Lin T, Botkin DJ, McCrum E, Winkler, H., and Norris, S.J.: Intact Flagellar Motor of
    Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring
    Curvature and Rotor/C Ring Assembly Flexion, J Bacteriol 191(16):5026-36, 2009.
  2. Sanabria H, Swulius MT, Kolodziej SJ, Liu, J., and Waxham NM: CaMKII Regulates Actin
    Assembly and Structure, J Biol Chem 284(15):9770-80, 2009.
  3. Liu, J., Howell, J.K., Bradley, S.D., Zheng, Y., Zhou, Z.H., and Norris, S.J.: Cellular architecture of Treponema pallidum: novel flagellum, periplasmic cone, and cell envelope as revealed by cryo electron tomography. J Mol Biol 403(4):546-61, 2010.
  4. Fu X, Walter MH, Paredes, A., ,Morais, M.C., and Liu, J.: The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography. Virology 421(2):141-8, 2011.
  5. Sze CW, Morado DR, Liu, J., Charon, N.W., Xu H, and Li, C.: Carbon storage regulator A (CsrA(Bb)) is a repressor of Borrelia burgdorferi flagellin protein FlaB. Mol Microbiol 82(4):851-64, 2011.
  6. Liu, J., Chen, C.Y., Shiomi. D,, Niki H, and Margolin, W.: Visualization of bacteriophage P1 infection by cryo-electron tomography of tiny Escherichia coli. Virology 417(2):304-11, 2011.
  7. Luther PK, Winkler, H., Taylor, K.A., Zoghbi ME, Craig R, Padrón R, Squire JM, and Liu, J.: Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci U S A 108(28):11423-8, 2011.
  8. Motaleb, M.A., Pitzer JE, Sultan SZ, and Liu, J.: A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi fliL mutant. J Bacteriol 193(13):3324-31, 2011.
  9. Xu H, Raddi G, Liu, J., Charon, N.W., and Li, C.: Chemoreceptors and flagellar motors are subterminally located in close proximity at the two cell poles in spirochetes. J Bacteriol 193(10):2652-6, 2011.
  10. Zhang K, Tong BA, Liu, J., and Li, C.: A single-domain FlgJ contributes to flagellar hook and filament formation in the Lyme disease spirochete Borrelia burgdorferi. J Bacteriol 194(4):866- 74, 2012.
  11. Zhang K, Liu, J., Tu Y, Xu H, Charon, N.W., and Li, C.: Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi. Mol Microbiol 85(4):782-94, 2012.
  12. Raddi G, Morado DR, Yan J, Haake DA, Yang XF, and Liu, J.: Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography. J Bacteriol 194(6):1299-306, 2012.
  13. Li, C., Kurniyati, Hu, B., Bian J, Sun J, Zhang W, Liu, J., Pan Y, and Li, C.: Abrogation of neuraminidase reduces biofilm formation, capsule biosynthesis, and virulence of Porphyromonas gingivalis. Infect Immun. 80(1):3-13, 2012.
  14. Liu, J., Hu, B., Morado DR, Jani S, Manson MD, and Margolin, W.: Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells. Proc Natl Acad Sci U S A 109(23):E1481-8, 2012.
  15. Hu, B., Margolin, W., Molineux, I.J., and Liu, J.: The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling during infection, Science 339(6119):576-9, 2013.
  16. Zhao, X., Zhang K, Boquoi T, Hu, B., Motaleb, M.A., Miller K, James M, Charon, N.W., Manson MD, Norris, S.J., Li, C., and Liu, J.: Cryo-Electron Tomography Reveals the Sequential Assembly of Bacterial Flagella in Borrelia burgdorferi. Proc Natl Acad Sci U S A, 110(35):14390-5, 2013.
  17. Chen, L., Xu, Q., Tu, J., Ge, Y., Liu, J., and Liang, F.T.: Increasing RpoS Expression Causes Cell Death in Borrelia burgdorferi. PLoS One 8(12):e83276, 2013.
  18. Hu, B., Morado DR, Margolin, W., Rohde JR, Arizmendi O, Picking WL, Picking WD, and Liu, J.: Visualization of the type III secretion sorting platform of Shigella flexneri. Proc Natl Acad Sci U S A 2015, 112(4):1047-1052. PMID: 25583506
  19. Lin T, Gao L, Zhao, X., Liu, J., and Norris, S.J.: Mutations in the Borrelia burgdorferi Flagellar Type III Secretion System Genes fliH and fliI Profoundly Affect Spirochete Flagellar Assembly, Morphology, Motility, Structure, and Cell Division. MBio. 2015 May 12;6(3). pii: e00579-15.
  20. Sultan SZ, Sekar P, Zhao, X., Manne A, Liu, J., Wooten RM, and Motaleb, M.A.: Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts. Infect Immun. 2015 May;83(5):1765-77.
  21. Yan R, Edwards TJ, Pankratz LM, Kuhn RJ, Lanman JK, Liu, J., and Jiang W: A fast cross- validation method for alignment of electron tomography images based on Beer-Lambert law. J Struct Biol. 2015 Oct 5. pii: S1047-8477(15)30082-4.
  22. Yan R, Edwards TJ, Pankratz LM, Kuhn RJ, Lanman JK, Liu, J., and Jiang W: Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer-Lambert law. J Struct Biol. 2015 Oct 7. pii: S1047-8477(15)30074-5.
  23. Hu, B., Margolin, W., Molineux, I.J., and Liu, J.: Structural remodeling of bacteriophage T4 and host membranes during infection initiation. Proc Natl Acad Sci U S A. 2015 Sep 1;112(35):E4919-28.
  24. Zhou, H.Z., McCarthy, D.B. O’Conner, C.M., Reed, L.J., and  Stoops, J. K. The remarkable structural and functional organization of the pyruvate dehydrogenase complexwe. Proc Natl Acad Sci USA Dec. 18; 98(26) (2001).