PMC

CryoEM analysis of parvovirus B19. (A) Resolution assessment of the 3D reconstructions of iB19, eB19 particles, and recombinant B19 VP2 VLPs (VP2 VLP). Correlation coefficients (black) and phase differences (gray) for different-resolution shells were computed from the structure factors derived from the independent reconstructions of two half data sets. These plots demonstrate that the data are reliable to at least 7.5-Å (iB19), 11.3-Å (eB19), and 7.7-Å (VP2 VLP) resolution based on a correlation coefficient of 0.5 (arrows) or a phase difference of 45°. (B) A stereodiagram of a density region of the iB19 cryoEM map congruent with the α-helical region of the B19 VP2 crystal structure (shown in red) validates the quality of the cryoEM density.

3D image reconstructions of parvovirus B19. (A) Stereoscopic view of a surface rendering of the iB19 particle at 7.5-Å resolution, viewed down an icosahedral twofold axis. The black triangular outline identifies an icosahedral asymmetric unit. The central cross-section is shown on the right, also viewed down an icosahedral twofold axis. Darker coloring in the central cross-section corresponds to higher electron density. The positions of icosahedral two-, three-, and fivefold axes are indicated. Structural differences between wild-type B19 and VP2 VLPs are located around the icosahedral fivefold axes. For one of the fivefold axes, densities near the viral surface, not present in VP2 VLPs, are highlighted with green arrows. (B) Image as described for panel A but illustrating the cryoEM density of eB19 particles at 11.3-Å resolution. (C) Image as described for panel A but showing the cryoEM density of recombinant B19 VP2 VLPs at 7.7-Å resolution. The equatorial slice is depicted at 7.7-Å (left) and 11.3-Å (right) resolution. One of the densities due to internally located VP2 termini pointing toward the center of the VP2 VLP is labeled with a blue arrow. The renderings are at sigma levels of 1.2, 1.5, and 2.0 for the maps in panels A, B, and C, respectively.

Significant differences between wild-type B19 and recombinant VP2 particles cluster at and around the icosahedral fivefold axes. (A) Stereoview of a surface rendering of a difference map between eB19 and B19 VP2 VLPs at 11.3-Å resolution, viewed down an icosahedral twofold axis. Positive densities are rendered in red, and negative densities in blue. The difference densities are superpositioned onto a semitransparent VP2 VLP. The black triangle marks an icosahedral asymmetric unit. On the right the central cross-section of the difference map is viewed down an icosahedral twofold axis. Black pixels represent positive density. The positions of icosahedral two-, three-, and fivefold axes are indicated. The most-significant positive differences (matter present in eB19 but not VLPs) are located around the fivefold cylindrical structure at the outer viral surface, labeled for one fivefold axis by red arrows, and within the fivefold channel, pointed out by green arrows. Significant negative difference densities (matter present in VLPs but not eB19) can be identified next to the fivefold axes at the inner viral surface (blue arrows). (B) Image as described for panel A but with a difference map between iB19 and B19 VP2 VLPs at 7.7-Å resolution. The largest positive differences besides the central DNA density are located around the fivefold cylinder at the outer viral surface, indicated by red arrows, and at the base of the fivefold channel, highlighted by green arrows. Connecting density diverges off the central fivefold axis halfway up the pore. (C) Image as described for panel A but the difference map is between iB19 and eB19 at 11.3-Å resolution. The most significant difference density is due to the presence of the DNA genome in infectious virions. In panel A, the renderings are at a sigma level of about 0.65 and 0.5 for positive and for negative density, respectively. The maps in panels B and C are rendered at a sigma level of about 0.6. The sigma values are based on the original maps, not on the difference maps.

Interpretation of difference densities between wild-type B19 and B19 VP2 VLPs. (A) Stereoview of the difference densities between eB19 and VP2 VLPs close to an icosahedral fivefold axis. Positive densities are rendered in red and negative densities in blue. The icosahedral axis is indicated by a gray rod. The fivefold proximal portions of one of five symmetry-related B19 VP2 molecules (PDB ID code 1S58) are shown as a ribbon diagram in half-transparent gold, while its N terminus is rendered nontransparent. Similarly, one MVM VP2 molecule (PDB ID code 1MVM) is shown in blue. Significant negative density (matter present in VLPs but not in eB19) is located at the inside of the virus particle and overlaps with the positions of the last ordered N-terminal residues in the crystal structure of B19 VP2 VLPs that point toward the viral center. The position of positive density at the base of the fivefold channel suggests that in eB19, the VP2 terminal sequence (residues 23 through 18, shown in pink) enters into the inner basal opening of the fivefold channel in a manner similar to that of the N-terminal residues of MVM. (B) Stereoview of the positive difference densities between iB19 and B19 VP2 VLPs close to an icosahedral fivefold axis. The difference map shows tubular density connecting the inside and outside of the viral particle. The Cα chain of B19 VP2 upstream of residue 18 was modeled into the difference density. The glycine-rich region (residues 17 through 12), shown in green, diverges from the central axis of the channel and connects with the difference density on the outside of the viral particle. The latter difference density could accommodate the remaining outermost N-terminal 11 residues of VP2, rendered in gold. (C) The VP2 N terminus (residues 1 through 24) of one VP, shown as described for panel B, is located between two symmetry-related VP2 molecules (gold and red). In contrast, the N-terminal residues 28 through 41 of MVM VP2, shown in blue, are positioned in the center of the fivefold channel (). (D) Organization of symmetry-related B19 VP2 termini (residues 1 through 24) at an icosahedral fivefold axis. The position of the B19 VP2 terminus makes it spatially possible for all VP termini to be externalized at the same time and closes the base of the fivefold channel.