Figure 1.
CryoEM and 3D reconstruction of hepatitis B virus (HBV) core assembled from full-length HBV core proteins at 3.5Å resolution.
(a, b) Representative focal-pair cryoEM images of core particles embedded in a thin layer of vitreous ice, recorded on Kodak So 163 film in an FEI Titan Krios cryo electron microscope operated at 300kV at liquid-nitrogen temperature. (c) Fourier transform of the cryoEM image in (a), showing contrast transfer function rings visible up to 1/3Å-1. (d) Shaded surface representation of the core reconstruction at 3.5Å resolution as viewed along a five-fold axis. One asymmetric unit is segmented and color-coded. The A, B, C, and D subunits that form the asymmetric unit are colored in red, green, yellow, and blue, respectively. The two-fold, three-fold, and five-fold axes are indicated by the numbers 2, 3, and 5, respectively. (e) Resolution assessment of HBV 3D reconstruction based on the 0.143 criterion of reference-based Fourier shell correlation coefficient, showing that the effective resolution is ~3.5Å. (f) Atomic model (stick) of an α-helix is superimposed in its density map (mesh). (g) An atomic model of an asymmetric unit of the T=4 HBV core derived from the cryoEM structure viewed along a five-fold axis. The A, B, C, and D subunits that form the asymmetric unit are color-coded in red, green, yellow, and blue, respectively.
Figure 2.
Comparisons between corresponding cryoEM structures (green) and crystal structures (red) by superimposition.
(a–d) The cryoEM-derived structures of the four subunits, namely subunits A (a), B (b), C (c), and D (d), from an asymmetric unit are similar to their crystal structure counterparts. Side chains are only shown for those residues that were resolved in the cryoEM structure of full-length core, but not in the crystal structure.
Figure 3.
The HBV core dimer structures and SDS-PAGE analysis of the HBV core protein.
(a) The dimer structure of subunits A (red) and B (green) shows no disulfide bond formation between the Cys61 residues of the two monomers (Insets). Density maps superimposed with atomic models of two helices from the boxed region, showing no density that can be attributed to disulfide bond formation between the two interacting monomers in this region. The density maps are contoured at 4.5σ (left inset) and 3σ (right inset) above the mean. (b) The dimer structure of subunits C (yellow) and B (blue) also show no disulfide bond formation between the Cys61 residues of the two monomers based on the cryoEM density map (Bottom insets). Density maps superimposed with atomic models of two helices from the boxed region, showing no density that can be attributed to disulfide bond formation between the two interacting monomers in this region. The density maps are contoured at 4.5σ (left inset) and 3σ (right inset) above the mean. (c) SDS-PAGE analysis of freshly-purified HBV core reveal no disulfide bond formation between core subunits. Lane 1: molecular-weight standards from Bio-Rad. Lane 2: HBV core boiled for 5 minutes under non-reducing condition; no dimer band was observed. Lane 3: HBV core boiled for 5 minutes under reducing condition with 5% (v/v) β-mercaptoethanol. A 10µg protein sample per lane was loaded and visualized by Coomassie Blue staining.
Figure 4.
Maps of HBV core reconstruction filtered to 10Å resolution.
All density maps are contoured at 2σ above the mean. (a) Inside view of the radially colored 3D reconstruction map, showing the full-length HBV core has a double-layer structure: an outer layer composed of the N-terminal core assembly domain (residues 1-149) and an inner layer composed of the basic C-terminal ARD (residues 150-185) and its bound RNAs. (b) A gray scale view of a central map slice from (a), showing links (arrows) connecting the outer layer and the inner layer. The links are located around 2-fold axes. (c) Density map superimposed with ribbon model of subunit C (yellow), showing the ordered C-terminal residues (residues 143-146) resolved in our cryoEM structure are superimposable with or close to the linker density around the 2-fold axis. The two-fold axis is indicated by the number 2. The ordered outer layer (transparent gray) is separated from the rest (green) of the structure by using atomic model. (d) A close-up view of density map, showing densities underneath one of the 12 vertices do not connect with either of the outer layer (gray) and the inner layer (green). Instead, these densities (arrow) extend toward the adjacent local 3-fold axes along the space between the outer and inner layers. The five-fold and local three-fold axes are indicated by the labels 5 and L3, respectively.
Figure 5.
Direct observation of the HBc C-terminal ARD segments protruding outside the core.
(a) Radially-colored, shaded surface representation of the HBV reconstruction filtered to 10Å resolution as viewed along a five-fold axis, showing some densities of the C-terminal ARD tails on the exterior of core (arrow). The two-fold, three-fold, local three-fold and five-fold axes are indicated by the labels 2, 3, L3, and 5, respectively. The density map is contoured at 1σ above the mean. (b) An enlarged view around a two-fold axis, showing a density ~20Å in length protruding outside the core through the local three-fold axis (arrow). The two-fold, three-fold, and local three-fold axes are indicated by the labels 2, 3, and L3, respectively. The ordered outer layer is colored in gray using atomic model, while the rest of density map is colored in green. The map is contoured at 1σ above the mean. (c) Density map (grey) superimposed with ribbon models of A (red) and B (blue) molecules, showing the positive-charged ARD density (green) interaction with the negative-charged D2 and E43 amino acids that line the local three-fold channel (L3). The ordered outer layer (transparent gray) is separated from the rest (green) of the structure by using atomic model. The density map is contoured at 1σ above the mean. (d, e) A slab (9.3 Å in thickness) across the local three-fold axis as viewed along a five-fold axis, showing densities (arrow) underneath one of the 12 vertices extending toward the adjacent local 3-fold axes and a possible pathway (red dotted line) for the ARD tail of molecule A protruding outside the core. While the ordered outer layer (gray) is contoured at 2σ above the mean in both maps, the rest (green) of the cryoEM map is contoured at 2σ in (d) and 1σ in (e), respectively. The five-fold and local three-fold axes are indicated by the labels 5 and L3, respectively.