Robert Duda

Research Assistant Professor

Phage & Virus Assembly

Office: (412) 624-4651
Lab: (412) 624-4651
A318 Langley Hall
4249 Fifth Avenue
Pittsburgh, PA 15260

Dr. Duda received his Ph.D. in 1983 with Fred Eiserling at the University of California, Los Angeles, performed his postdoctoral studies with Roger Hendrix at the University of Pittsburgh, and joined the Department in 2000.

Our lab uses bacteriophages as model systems to learn how proteins assemble into complex biological structures. The structural components of phages are particularly appropriate because they are large enough that we can visualize them using electron microscopy and detect them using simple gel assays, but simple enough that we know all of the genes required to produce them and can easily make mutations and study their effects. Three easily studied parts of phages are tails, tail fibers, and capsids. My research focuses mainly on capsids and in particular the capsid of E. coli phage HK97 and some close relatives.

 Viral capsids are superficially simple: they are made from a small number of proteins that make up the capsid shell as hexons on the capsid faces and pentons at the corners. In many cases there are proteins or domains required for assembly, but missing from the final structure - these serve as chaperones or scaffolding proteins.

HK97 Capsid Assembly Pathway

              (above) The Bacteriophage HK97 Capsid Assembly Pathway

As a model system, the HK97 capsid has many advantages, 1) it is simple - only three genes are required to build it, 2) it is efficiently made in the laboratory using plasmid expression of cloned genes, 3) much of the pathway of its assembly and maturation are well understood, and 4) there is a large repertoire of structural information from both electron microscopy and x-ray crystallography.

Research topics in the lab:

How does the same protein assemble into hexamers and pentamers?

How is capsid size determined?

How is the capsid maturation protease incorporated and regulated?

How is the portal incorporated into one and only one vertex? - The portal forms the site where DNA goes in and out of the capsid and where the tail is attached.

What are the functions of the HK97 scaffolding-protein-like domain called the delta domain? - This domain is comprised of the first ~100 residues of the major capsid protein and is required for assembly, but removed by proteolysis after assembly is complete.

Comparative capsidomics: We are also studying two larger phages, called D3 and phi1026b in collaboration with James Conway (here in Pittsburgh). These two phages use proteins which are very similar to make capsids that have ~540 subunits in an arrangement called T=9 vs the 420 subunits used by HK97 to make its smaller T=7 capsids. We are hoping that sub-nanometer electron density maps of these larger capsids will help us build models that suggest how capsid size is determined.

Fitting Capsid model into electron density map

(above) Cryo-EM Density Map of  D3 Prohead fit with Models based on a HK97 Prohead II x-ray structure

Opportunities for undergraduates: In the past few years we have developed new "mini" capsid production methods that have made it easier for undergraduates to participate in our research program. Several dedicated undergraduate researchers have made significant contributions to our work by making capsid gene mutations on plasmids and rapidly characterizing how the mutations affect capsid assembly.

Collaboration: My main collaborators are Roger Hendrix in Biological Sciences (with whom I share a large, well-equipped lab), James Conway in the Structural Biology Dept. at the University of Pittsburgh Medical School, Alasdair Steven and Naiqian Cheng in the Laboratory of Structural Biology at the NIH and Jack Johnson at The Scripps Research Institute in La Jolla, CA.

Recent Publications
  • Tso DJ, Hendrix RW, Duda RL (2014) Transient contacts on the exterior of the HK97 procapsid that are essential for capsid assembly. J Mol Biol. 426:2112-2129

  • Cardone G, Duda RL, Cheng N, You L, Conway JF, Hendrix RW, Steven AC. (2014) Metastable intermediates as stepping stones on the maturation pathways of viral capsids. MBio.5:e02067-14

  • Oh B, Hendrix RW, Duda RL (2014) The delta domain of the HK97 major capsid protein is essential for assembly. Virology 456-457:171-178

  • Xu, J., R. W. Hendrix, and R. L. Duda (2014) Chaperone-Protein Interactions That Mediate Assembly of the Bacteriophage Lambda Tail to the Correct Length. J. Mol. Biol. 426: 1004-1018.

  • Xu, J., R. W. Hendrix, and R. L. Duda (2013) A Balanced Ratio of Proteins from Gene G and Frameshift-Extended Gene GT Is Required for Phage Lambda Tail Assembly. J. Mol. Biol. 425:3476-87

  • Duda, RL, Oh B, Hendrix RW. (2013) Functional domains of the HK97 capsid maturation protease and the mechanisms of protein encapsidation. J Mol Biol. 2013 425:2765-81

  • Huang, R.K., R. Khayat, K.K. Lee, I. Gertsman, R.L. Duda, R.W. Hendrix, and J.E. Johnson (2011) The Prohead-I Structure of Bacteriophage HK97: Implications for Scaffold-Mediated Control of Particle Assembly and Maturation. J Mol Biol 408:541-554.

  • Dierkes, L.E., C.L. Peebles, B.A. Firek, R.W. Hendrix, and R.L. Duda (2009) Mutational analysis of a conserved glutamic acid required for self-catalyzed cross-linking of bacteriophage HK97 capsids. J. Virol. 83:2088-2098

  • Duda, R.L., P.D. Ross, N. Cheng, B.A. Firek, R.W. Hendrix, J.F. Conway, and A.C. Steven (2009) Structure and energetics of encapsidated DNA in bacteriophage HK97 studied by scanning calorimetry and cryo-electron microscopy. J. Mol. Biol. 391:471-483

  • Gertsman, I., L. Gan, M. Guttman, K. Lee, J.A. Speir, R.L. Duda, R.W. Hendrix, E.A. Komives, and J.E. Johnson (2009) An unexpected twist in viral capsid maturation. Nature 328:229-240

  • Conway, J.F., N. Cheng, P.D. Ross, R.W. Hendrix, R.L. Duda, and A.C. Steven (2007) A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged. J. Struct. Biol. 158:224-232

  • Xu, J., R. W. Hendrix, and R. L. Duda. 2004. Conserved translational frameshift in dsDNA bacteriophage tail assembly genes. Mol Cell 16:11-21.

  • Helgstrand, C., W. R. Wikoff, R. L. Duda, R. W. Hendrix, J. E. Johnson, and L. Liljas. (2003). The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution. J Mol Biol 334:885-99.

  • Conway, J. F., W. R. Wikoff, N. Cheng, R. L. Duda, R. W. Hendrix, J. E. Johnson, and A. C. Steven. (2001) Virus maturation involving large subunit rotations and local refolding. Science 292:744-8.

  • Lata, R., J. F. Conway, N. Cheng, R. L. Duda, R. W. Hendrix, W. R. Wikoff, J. E. Johnson, H. Tsuruta, and A. C. Steven (2000) Maturation dynamics of a viral capsid: visualization of transitional intermediate states. Cell 100:253-63.

  • Wikoff, W. R., L. Liljas, R. L. Duda, H. Tsuruta, R. W. Hendrix, and J. E. Johnson (2000) Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289:2129-33.

  • Duda, R. L. (1998) Protein chainmail: catenated protein in viral capsids. Cell 94:55-60.

  • Conway, J. F., R. L. Duda, N. Cheng, R. W. Hendrix, and A. C. Steven (1995) Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system. J Mol Biol 253:86-99

  • Duda, R. L., J. Hempel, H. Michel, J. Shabanowitz, D. Hunt, and R. W. Hendrix (1995) Structural transitions during bacteriophage HK97 head assembly. J Mol Biol 247:618-35.

  • Duda, R.L., 2008. Icosahedral Tailed dsDNA Bacterial Viruses. Encyclopedia of Virology 2008, 30-37.