J. Mol. Biol. (1996) 260, 85–98 Three-dimensional Structure of Scaffolding-containing Phage P22 Procapsids by Electron Cryo-microscopy Pamela A. Thuman-Commike 1,2 , Barrie Greene 4 , Joanita Jakana 3 B. V. Venkataram Prasad 1,2,3 , Jonathan King 4 , Peter E. Prevelige Jr 5 and Wah Chiu 1,2,3 * 1 Program in Structural & The procapsids of bacterial viruses are the products of the polymerization Computational Biology & of coat and scaffolding subunits, as well as the precursors in DNA packaging. Electron cryo-microscopy has been used to study the Molecular Biophysics 2 The W. M. Keck Center for three-dimensional structures of bacteriophage P22 procapsids containing wild-type and mutant scaffolding proteins. The scaffolding mutant Computational Biology and 3 Verna and Marrs McLean structure has been resolved to 19 Å resolution and agrees with the 22 Å resolution wild-type procapsid reconstruction. Both procapsid reconstruc- Department of Biochemistry Baylor College of Medicine tions contain an outer icosahedral coat protein shell and an inner One Baylor Plaza, Houston scaffolding protein core. The outer core protein forms a T = 7 icosahedral TX 77030, USA lattice with distinctive channels present at the centers of the pentons and hexons. In addition, the hexons display a prominent skew. Computational 4 Department of Biology isolation of the skewed hexon shows the presence of a local 2-fold axis that Massachusetts Institute of reduces the number of unique conformations in the asymmetric unit to Technology, Cambridge, MA four at this resolution. We have classified the four unique subunits into 02142, USA three distinct classes, based upon the shape of the upper domain and the presence of a channel leading to the inner coat protein surface. In addition, 5 Department of Microbiology at the inner surface of the coat protein, finger-like regions that extend University of Alabama at towards the scaffolding protein core are present in two of the subunits. The Birmingham, Birmingham finger-like regions suggest the presence of an ordered interaction between AL 35294, USA the inner coat protein and the scaffolding protein. However, an icosahedral scaffolding protein shell is not formed, and the innermost scaffolding protein core does not pack with icosahedral symmetry.  1996 Academic Press Limited Keywords: bacteriophage P22; electron cryo-microscopy; image *Corresponding author processing; three-dimensional structure; virus assembly Introduction A common pathway in the formation of a double-stranded (ds) DNA-containing icosahedral virion is the assembly of a procapsid lacking the complete genome. Typically, such procapsids are assembled with the assistance of scaffolding proteins, which are absent from the mature virion. This assembly pathway is found in dsDNA bacteriophages including Salmonella typhimurium phage P22, the Escherichia coli phages T7, T3, T4 and , and Bacillus subtilis phage 29 (Casjens & Hendrix, 1988). In addition, the single-stranded DNA phage X174 utilizes such a procapsid (Hayashi et al ., 1988). Scaffolding protein assisted assembly is not unique to bacteriophages. The first product of the herpes simplex virus assembly pathway is a non-DNA-containing B capsid (O’Callaghan et al ., 1977; Newcomb & Brown, 1991; Rixon, 1993) with an internal scaffolding core that is not present in the mature virion. A similar assembly intermediate has been identified for adenovirus (Edvardsson et al ., 1976; D’Halluin et al ., 1978). The adenovirus intermediate contains two proteins thought to be scaffolding proteins that are not present in the mature virion (Hasson et al ., 1989, 1992). Scaffolding proteins can function as internal or external frameworks for assembly of coat lattices. Recent structural studies have revealed the ar- rangement of external scaffolding proteins on procapsids from two different phages (Ilag et al ., 1995; Marvik et al ., 1995). However, the organiz- ation of an internal scaffolding protein within an assembled procapsid has not been determined for any prokaryotic or eukaryotic virus. In particular, Abbreviation used: ds, double-stranded. 0022–2836/96/260085–14 $18.00/0  1996 Academic Press Limited