Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Angstrom, enabling direct visualization of alpha-helical secondary structure. To help correlate such density maps wit h the amino acid sequences of the component proteins, we advocate peptide-b ased difference mapping, i.e., insertion of peptides, approximate to 10 res idues long, at targeted points in the sequence and visualization of these p eptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Angstrom resolution Hepatit is B virus capsids are icosahedral particles, approximate to 300 Angstrom i n diameter, made up of T-shaped dimers (subunit M-r, 16-21 kDa, depending o n construct). The stems of the Ts protrude outward as spikes, whereas the c rosspieces pack to form the contiguous shell. The two N termini per dimer r eside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecu lar disulfide bond between the cysteines at positions 61 and -7 (in the res idual propeptide) in the "e-antigen" form of the capsid protein and has imp lications for why this clinically important antigen remains unassembled in vivo.