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: 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 |
|
High resolution structures of Flaviviruses
| Many flaviviruses, such as West Nile virus, dengue virus, Japanese encephalitis virus, and yellow fever virus, are arthropod-borne human pathogens. These viruses are of global concern for major epidemics that can cause millions of infections and many deaths each year. Currently, there is no commercially available vaccine, and treatment relies on supportive therapy. The flavivirus mature particles are small enveloped viruses with a diameter of about 50 nm. The infection starts with the mature virus particles binding to the host surface receptor. The particles then enter the host cell via receptor-mediated endocytosis. The low pH in the endosome triggers the fusion of viral membrane with the endosomal membrane and the release of the ssRNA viral genome into the cytoplasm. The ~11 kb genome has a single long open reading frame that encodes a large polyprotein. The polyprotein is co- and post-translationally cleaved by host and virus-encoded proteases into at least ten proteins. The three structural proteins (C, capsid protein; prM precursor of protein M, and E protein) are located at the N terminus while the rest are for the seven non-structural proteins. The virus particles assemble at the endoplasmic reticulum (ER) membrane when capsid proteins complex with the viral genome, bud into the lumen of ER, and associate with the prM and E proteins to form the immature particles. The immature particles are transported in the exocytic pathway where the prM protein is cleaved by the host furin-like protease in the trans-Golgi network. The cleavage of prM and dissociation of pr peptide from the particle induce dramatic rearrangement and maturation of the particle. The mature particles are subsequently released. While there have been extensive studies of the structure of many flaviviruses, there is still no atomic resolution structure of the entire flavivirus particle. To better understand the structural basis of flavivirus assembly and infection, we are using cryo-EM imaging and 3-D reconstruction method to solve the near-atomic resolution (~4Å) structure of flavivirus at both the immature and the mature states. This project is in collaboration with the group of Michael Rossmann and the group of Richard Kuhn. |
|
Determination of membrane protein structure by 2-D crystallization and electron crystallography
|
Although about a quarter of the entire genome encode membrane proteins, the number of structures of membrane protein is very small compared to the explosion of the number of solubule protein structures. Instead of pursuing X-ray crystallography of 3-D crystals of membrane proteins grown in detergents, we aim to solve membrane protein structures in their native lipid bilayer environment by using electron crystallography of 2-D crystals. The success of electron crystallography is criticially dependent on the order of the 2-D crystals just like it for X-ray crystallography of 3-D crystals. The current crystallization pipeline, which consists of detergent solubulization, purification, reconstiution into liposome by dialysis, and nagative staining TEM checking for crystal, is time consuming and requires large amount of samples per screened condition. These limitations make it extremely hard to screen large number of conditions as is routinely done for 3-D crystallization. We are interested in exploring alternative approaches to circumvent these limitations and to allow much more straightforward 2-D crystallization of membrane proteins. |
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: the 4.5 Å resolution structure of bacteriophage Epsilon15. The reconstruction allowed the construction of backbone model for its major shell protein gp7. We have since further developed the image processing method and improved the reconstruction to 3.7 Å resolution based on Fourier Shell Correlation (0.5 threshold criterion) of two reconstructions from half datasets without additional non-crystallographic symmetry averaging (unpublished). We are interested in further pushing the resolution to 3 Å and beyond. It is likely that improvements should be needed in every aspect of a cryo-EM project: TEM microscope, sample preparation, image collection, 2-D alignment, CTF estimation and correction, 3-D reconstruction, and large scale computing. |
|
|
References: 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 |
|