Psittacine beak and feather disease results in the irreversible loss of feathers and deformities in the beak and claws of parrots. The etiological agent of the disease is the beak and feather disease virus (BFDV), a member of the Circoviridae family. BFDV is an un-enveloped, T=1 icosahedral virus, and is one of the smallest known viruses, <20 nm in diameter. The circular 2,000 bp ssDNA genome encodes only two proteins: the replicase (Rep) and the capsid protein (Cap). Cap is multifunctional and facilitates viral attachment and entry into cells, uncoating of the genome, and assembly of new particles.
Recently a structure of an icosahedral virus-like particle (VLP) formed by the Cap protein bound to ssDNA was determined by X-ray crystallography at a resolution of 2.5Å (1). There are several highly positively charged domains in the N-terminus of Cap which are thought to be responsible for binding to the ssDNA genome and triggering assembly. However, most of the N-terminus of the protein subunit and ssDNA are not visible in the crystal structure. Using a complementary structural method, cryogenic-electron microscopy (cryoEM), allowed us to resolve some of the missing features in the crystal structure.
A total of 1,288 micrographs were collected on a 300kV microscope equipped with a direct electron detector. Using the MASSIVE supercomputer cluster, 2D and 3D class averages were generated from a dataset of 22,663 particles. The particle orientations were refined in RELION using the gold standard approach to generate a 4.5Å map of the VLP. By comparing the map to the crystal structure, we have identified electron density belonging to the ssDNA and several positively-charged residues that form the DNA-binding ‘footprint’ in BFDV. These studies further our understanding of viral assembly and illustrate the complementarity of cryoEM and X-ray crystallography studies.