PMC

Agarose gel showing the presence of DNA in VLPs. Lane 1, molecular weight marker (500 bp band labeled); Lane 2 untreated (no DNAse or RNAse); Lane 3 treated with RNAse; and Lane 4 treated with DNAse.

Cryo-reconstructions of AAV2 VLPs and AAV2-Hep complex. (A–D) Shaded-surface representations of VLP reconstruction showing an entire particle viewed along an icosahedral twofold axis (A) and close-up views along two- (B), three- (C), and five-fold (D) axes. (E–H) Same as (A–D) for AAV2-Hep complex. The top of one βDE ribbon is marked with a black dot in D and H to visualize the ~36° difference in the position of this loop in the two maps. Broken circles (B, C, F, and G) highlight regions of obvious difference in the two reconstructions. These images were generated in RobEM ().

Difference density associated with the interior of the AAV2 capsid. (A) The positive difference density at 1σ (solid red) and 0.5σ (red mesh) superimposed onto a shaded-surface representation of the AAV2 crystal structure (solid grey). (B) The dodecahedral cage at 1σ (red mesh) superimposed with the nucleotides (NTs) ordered in the crystal structures of AAV4 (blue) and MVMi (cyan). (C) Close-up of Cα chains (ribbons and coil) and the side-chains (stick) of AV2 residues (in a trimer) that closely associate with the AAV4 (blue) and the MVMi (cyan) NTs. The amino acids at a radial distance of <3.5 Å (D237, R238, R307, F420, G638, E685) and <5.0 Å (H627, F628, H629, P630) from the modeled NTs are indicated with black arrows for one VP monomer. The two- and threefold axes are indicated with numbers in (A and B) and the threefold axis is indicated with a filled triangle in (C). (A and C) are viewed down a threefold axis, and (B) down a twofold axis. The figure was generated using Chimera ().

Capsid conformational transitions at the fivefold axis. Equatorial cross-sections of the (A) VLP and (B) AAV2-Hep reconstructions. (C) Difference map (AAV2-Hep minus AAV2 VLP), with positive (red) and negative (cyan) differences superimposed onto a shaded-surface density map (grey mesh) of the AAV2 crystal structure close to the fivefold axis. Positive and negative density differences are labeled (e.g. 2f pos = positive density close twofold axis). (D) Close-up of fivefold channel and HI loop difference densities. HI loop is shown as observed in the crystal structure (cyan coil) close to the negative (cyan) density and modeled (magenta coil) into positive (red) density. βDE ribbons (cyan coil, as in the crystal structure) are indicated with arrows. (E) Top-down view of HI loop model. Positions of residues K321 and E322 at the base of the βDE ribbon, and Y673 at the base of the HI loop are indicated by arrows for some of the monomers. The cyan loops and residues indicate the position of these regions in the crystal structure. The magenta coil indicates proposed new position of HI loop. (F) Side-view of HI loop model. The positions of the HI loops when heparin is not bound (cyan) and when heparin is bound (magenta). Residue D327 at the tip of the βDE ribbon, and the HI loop are indicated with arrows for one monomer. A two-, three-, and fivefold axis are indicated on each panel where appropriate. Figure (A) was generated using RobEM () and (C–F) by Chimera ().

AAV2 crystal structure with superimposed AAV2-Hep minus AAV2 VLP positive difference map and heparin models. A) Shaded-surface electron density map of AAV2 (PDB accession No. 1LP3, ) (grey) with the positive density (red mesh, contoured at 1σ) close to the two-, three-, and fivefold axes, 2f pos, 3f pos and 5f pos1, respectively, superimposed. (B) Close-up of shaded-surface representation of VLP reconstruction (grey) and AAV2-Hep (pink, density not in present VLPs), with the pseudo-atomic coordinates of heparin oligosaccharides (solid red) modeled into positive density (red mesh). hep2f = decasaccahride (twofold symmetry related decasaccahride is also shown); hep3f = single disaccharide (threefold symmetry related models are also shown). (C) Ribbon diagram of an AAV2 VP3 trimer and modeled heparin molecules. Surface heparin binding residues, R484, K527, K532, R585, and R588, are shown in blue (Arginines) or light blue (lysines) with terminal amino groups depicted as black balls. Residues from a reference monomer are denoted with A after the residue number; B indicates a threefold related residue. Heparin models are as labeled in (B). (D) Close up of basic (blue), acidic (orange) residues flanking the heparin binding residues. Basic residues are colored as in (C) and carboxyl groups are white for acidic residues. The broken lines indicate potential ionic interactions. The location of the icosahedral symmetry axes are labeled with numbers in (A–C). (A) is viewed along a twofold axis, and B, C, and D along a threefold axis. The figure was generated using Chimera ().