Viral connectors are essential components of the DNA packaging machinery. T
hey interact with nucleic acids and other viral components to translocate D
NA inside the viral head. We have attempted to locate the different structu
ral and functional domains of the phage Phi 29 connector using a combinatio
n of approaches to generate different antigenic probes. Complexes of native
connectors with either monoclonal or monospecific antibodies were studied
by immunoelectron microscopy and image averaging methods. The data were mer
ged in a model of the connector domain structure at 2-3 nm resolution. This
epitope mapping provides a general outline of the folding architecture of
the connector polypeptide, following a complicated threading that places th
e amino and carboxyl-terminals in close alignment in the narrower domain at
2-3 nm from the top of the connector. The appendages are built up by a lon
g and highly immunogenic sequence (amino acid residues 153 to 206). The RNA
binding domain forms part of the top of the narrow conical area of the con
nector, a flexible region that undergoes structural changes during viral mo
rphogenesis. The DNA binding domain is located not far away, 2-3 nm below,
in the outer side of the narrow conical part. The precise location of the f
unctional domains of the connector, as well as their relative positions pro
vide the first experimental framework for understanding the connector funct
ion. (C) 1999 Academic Press.