Structural basis of tailed dsDNA bacteriophage assembly and DNA packaging

The majority of viruses infecting bacteria are of the tailed dsDNA variety. It is estimated that 1031 tailed dsDNA bacteriophages exist and thus are likely to be the most abundant life forms in the biosphere. These tailed dsDNA phages share a common morphogenetic strategy beginning with the assembly of a precursor capsid, or procapsid, which is formed by the copolymerization of capsid and scaffolding proteins. The procapsid acts as a preformed container into which the viral DNA is subsequently pumped, and out of which the scaffolding protein must exit. DNA enters the procapsid through a dodecameric portal protein ring located at a single vertex within the capsid protein lattice. This portal protein ring breaks the symmetry of the icosahedral capsid and thus its location is often referred to as the unique vertex. Together with the portal protein, DNA packaging is driven by the terminase, a protein complex composed of a large and a small subunit. Terminase cuts the concatemeric replicative DNA after an appropriate length has been packaged. The packaging of DNA needs to overcome substantial resistance against the pressure inside the capsid (estimated to be about 10x of that in a champaign bottle). After DNA packaging, tailhub and tailspikes tightly bind the portal at the unique vertex to form the infectious particle. During infection, the binding of tailspikes on host cell surface will trigger the release of the packaged DNA from the phage capsid and injection into host cell. The phage capsid remains outside of the host cell. Though the above overall pathway is known for decades, the detailed mechanisms for the assembly, DNA packaing, and infection are still largely uncertain. We are interested in understanding these processes using a structural approach, primarily with cryo-EM imaging and 3-D reconstruction.
References:
Baker, M.L., Hryc, C.F., Zhang, Q., Wu, W., Jakana, J., Haase-Pettingell, C., Afonine, P.V., Adams, P.D., King, J.A., Jiang, W. & Chiu, W. (2013). Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling. Proceedings of the National Academy of Sciences 110:12301–12306. PubMed|PDF
Guo, F., Liu, Z., Vago, F., Ren, Y., Wu, W., Wright, E.T., Serwer, P. & Jiang, W. (2013). Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7. Proceedings of the National Academy of Sciences 110:6811–6816. PubMed|PDF
Jiang, W., Baker, M. L., Jakana, J., Weigele, P., King, J. and Chiu, W. 2008. Backbone Structure of the Infectious Epsilon15 Virus Capsid Revealed by Electron Cryomicroscopy. Nature 451(7182):1130-4. PubMed|PDF
Chang, J., Weigele, P., King, J., Chiu, W., and Jiang, W. 2006. Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure 14(6):1073-82. PubMed|PDF
Jiang, W., Chang, J., Jakana, J., Weigele, P., King, J., and Chiu, W. 2006. Structure of complete Epsilon15 phage reveals organization of condensed DNA and DNA packaging/injection apparatus. Nature 439(7076):612-616. PubMed|PDF
Jiang, W., Li, Z., Zhang, Z., Baker, M. L., Prevelige, P. E., and Chiu, W. 2003. Coat protein fold and maturation transition of bacteriophage p22 seen at subnanometer resolutions. Nat. Struct. Biol. 10:131-135. PubMed|PDF


Near-atomic resolution (3-4 Å) single particle cryo-EM: instruments, image processing algorithms, and large scale computing

Understanding of the functional mechanisms of macromolecules can be significantly enhanced by the determination of their structures at high resolution and the availability of atomic models. X-ray crystallography has been extremely successful in solving numerous atomic structures. However, structures of many large macromolecular complexes in the cell and large viruses remain to be determined due to difficulty in obtaining diffracting crystals. Single particle cryo-electron microscopy and 3-D reconstruction (cryo-EM), without the need of crystal, has emerged as a promising method that is well suited to determine the structures of large macromolecular complexes and viruses. It is now relatively routine to achieve subnanometer resolutions (6-10 Å). We have now demonstrated a reconstruction at near-atomic resolution (based on gold standard FSC=0.143 criterion): the 4.5 Å resolution structure of bacteriophage Epsilon15, 3.5 Å resolution structure of bacteriophage T7, and 2.95 Å resolution structure of the smallest virus circovirus. The reconstruction allowed the construction of atomic models for the major shell proteins. We are interested in further pushing the resolution to 2.5 Å and beyond to resolve finer structural details sufficient for drug design. It is likely that improvements should be needed in every aspect of a cryo-EM project: TEM microscope, camera, sample preparation, image collection, 2-D alignment, CTF estimation and correction, 3-D reconstruction, and large scale computing.
References:
Guo, F. & Jiang, W. (2014). Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses. Methods Mol Biol 1117:401–443. PubMed|PDF
Baker, M.L., Hryc, C.F., Zhang, Q., Wu, W., Jakana, J., Haase-Pettingell, C., Afonine, P.V., Adams, P.D., King, J.A., Jiang, W. & Chiu, W. (2013). Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling. Proceedings of the National Academy of Sciences 110:12301–12306. PubMed|PDF
Jiang, W., Guo, F. & Liu, Z. (2012). A graph theory method for determination of cryo-EM image focuses. J Struct Biol 180:343–352. PubMed|PDF
Chen, D.-H., Baker, M.L., Hryc, C.F., Dimaio, F., Jakana, J., Wu, W., Dougherty, M., Haase-Pettingell, C., Schmid, M.F., Jiang, W., Baker, D., King, J.A. & Chiu, W. (2011). Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus. Proceedings of the National Academy of Sciences 108:1355–1360. PubMed|PDF
Jiang, W., Baker, M. L., Jakana, J., Weigele, P., King, J. and Chiu, W. 2008. Backbone Structure of the Infectious Epsilon15 Virus Capsid Revealed by Electron Cryomicroscopy. Nature 451(7182):1130-4. PubMed|PDF
Liu, X., Jiang, W., Jakana, J. and Chiu, W. 2007. Averaging tens to hundreds of icosahedral particle images to resolve protein secondary structure elements using a multi-path simulated annealing optimization algorithm. J. Struct. Biol. 160(1):11-27, PubMed|PDF
Jiang, W., Li, Z., Zhang, Z., Booth, C. R., Baker, M. L., and Chiu, W. 2001. Semi-automated icosahedral particle reconstruction at sub-nanometer resolution. J. Struct. Biol. 136:214-225, PubMed|PDF


3-D structure of DNA nano-particles

DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute molecular computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large numbers (hundreds) of unique DNA strands poses a challenging design problem. Our collaborator Prof. Chengde Mao group has demonstrated a simple solution, mimicking the self assembly principle of biological viruses, to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger three-dimensional structures. We test this hierarchical self-assembly concept with DNA molecules that form three-point-star tiles and five-point-star tiles. By controlling the flexibility and concentration of the tiles, the one-pot assembly yields tetrahedra, dodecahedra, buckyballs, or icosahedra that are tens of nanometres in size. The 3-D structures of these nano-particles were imaged and reconstructed using cryo-EM. The assembly strategy can potentially be adapted to allow the fabrication of a range of relatively complex three-dimensional structures.
References:
Zhang C, Su M, He Y, Zhao X, Fang PA, Ribbe AE, Jiang W, Mao C. 2008. Conformational flexibility facilitates self-assembly of complex DNA nanostructures. Proc Natl Acad Sci U S A.105(31):10665-9, PubMed|PDF
He, Y., Ye, T., Su, M., Zhang, C., Ribbe, A. E., Jiang, W. and Mao, C. 2008. Hierarchical Self-Assembly of Symmetric Supramolecular Polyhedra. Nature 452(7184):198-201, PubMed|PDF