FIG 3.

Biochemical characterization and 3D cryo-EM reconstructions of IBDV E1 capsids. (A to C) The E1 pooled preparation was ultracentrifuged through a sucrose gradient, and 12 fractions were collected, concentrated, analyzed by SDS-PAGE, and developed by Coomassie blue staining (A) or by Western blotting using anti-VP2 (B) and anti-VP3 (C) antibodies. The direction of sedimentation was from right to left, with fraction 12 being at the gradient top. Molecular size markers (10³ Da) are indicated at the left; the pVP2, VP2, and VP3 bands are indicated. (D) Electron microscopy of negatively stained E1 particles. (Left) Purified E1 particles after ultracentrifugation in a CsCl gradient; (center) T=13 capsid-enriched E1 fraction obtained after consecutive ultracentrifugation in a sucrose gradient, corresponding to central fractions (fractions 6 and 7); (right) T=1 capsid-enriched E1 fraction obtained after consecutive ultracentrifugation in a sucrose gradient, corresponding to upper fractions (fractions 10 and 11). (E) Cryo-electron micrograph of twice-purified E1 T=13 capsids from fractions 6 and 7 in panels A and D. Red arrows indicate E1-1 particles whose morphology is characterized by a thickened capsid wall with an empty central region; blue arrows indicate E1-2 particles that are homogeneously dark. Bar, 100 nm. (F) Radial density profiles from averaged 3D maps of E1-1A, E1-1B, and E1-2 particles. A radial profile calculated from a previous IBDV map is included as a control. Note that the VP3 layer (centered at an ∼225-Å radius), as well as the Fresnel ring at the inner capsid surface (at an ∼250-Å radius), decreases from E1-1A to E1-1B to E1-2. The E1-1A and E1-1B capsids are completely empty in the central region (to an ∼150-Å radius). (G) Radially color-coded inner (top) and outer (bottom) surfaces of E1-1A (left), E1-1B (center), and E1-2 capsids (right) viewed along an icosahedral 2-fold axis (yellow, T=13 shell; orange, VP3 density). Maps are calculated with (left) and without (right) icosahedral symmetry and are contoured at 1.25 σ above the mean density. Bar, 100 Å.