Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

Abstract

Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication.

Figure 1

AAV-3b crystals were grown in 3.4% PEG 6000 in HM buffer by vapor diffusion. Corner to corner, the maximum dimension of this crystal is approximately 0.4 mm.

Figure 2

Diffraction image from AAV-3b crystals which diffract X-rays to 3.0 Å resolution. A dual-detector system was used to maximize coverage of the diffraction pattern, while maintaining adequate spot separation, and the diffraction pattern from one of the detectors is shown.

Figure 3

Rotation functions of AAV-3b. Ordinary self-rotation functions for (a) 3.0 Å and (c) 2.6 Å resolution data sets, calculated at 12 and 9.3 Å, respectively. The panels show spherical polar projections oriented with the hexagonal setting of the rhombohedral lattice (a, b*, c*) as indicated. (a) The ordinary self-rotation function (RF) at κ = 72°, calculated with the 13% complete untwinned data set is imperfect, but shows the locations of the six expected icosahedral fivefolds (shown as pentagons). (b) In spite of the truncation errors evident in the ordinary RF, when icosahedral symmetry is imposed as a constraint in a 14 Å locked RF, an unambiguous solution for the icosahedral orientation emerges. (c) The additional symmetry in the 2.6 Å resolution data is a result of twinning. Fivefold peaks are observed from both twin domains and are highlighted as black and gray hexagons.

Figure 4

Initial averaged electron-density maps. Maps were created by phase extension from 3.75 to 3.0 Å, averaging 20 subunits related by NCS. Starting phases were taken from (a) AAV-4 (55% sequence identity to AAV-3B for VP3) or (b) AAV-2 (87% identity). Overlaid is the preliminary model of AAV-3b. The region shown was omitted from the phasing model. Recovery of density for the omitted region shows that the power of 20-fold NCS averaging is sufficient to recover independent phase information, even with the 13% of the data that were from the untwinned crystal. With the AAV-2 phasing model, the density was of sufficient quality to remodel the regions that differed. Remodeling using density from the AAV-4 phasing model would have been more challenging, but might have been possible in several rounds of bootstrapping.