Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 7

Abstract

Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids diffract X-rays to approximately 3.0 A resolution. The crystals belong to the rhombohedral space group R3, with unit-cell parameters a = 252.4, c = 591.2 A in the hexagonal setting. The diffraction data were processed and reduced to an overall completeness of 79.0% and an R(merge) of 12.0%. There are three viral capsids in the unit cell. The icosahedral threefold axis is coincident with the crystallographic threefold axis, resulting in one third of a capsid (20 monomers) per crystallographic asymmetric unit. The orientation of the viral capsid has been determined by rotation-function searches and is positioned at (0, 0, 0) by packing considerations.

Figure 1

Purification and crystallization of AAV7 viral capsids. (a) SDS–PAGE gel showing the AAV7 viral proteins VP2 and VP3 (molecular weights 73 and 62 kDa, respectively) assembled into capsids in the baculovirus expression system in the right lane. VP1 was not present in the capsids. The left lane shows the position of molecular-weight standards (kDa). (b) Transmission electron micrograph of intact AAV7 capsids stained with 2% uranyl acetate. (c) Optical photograph of an AAV7 capsid crystal.

Figure 2

X-ray diffraction image of an AAV7 crystal: image of a typical 0.3° oscillation photograph. The inner and outer concentric rings indicate the 5.0 and 3.2 Å resolution shells, respectively.

Figure 3

AAV7 viral capsid orientation in the H3 hexagonal crystal unit-cell setting. The red and blue contours show the fivefold and threefold icosahedral symmetry elements, respectively, for self-rotation function searches with κ = 72° and 120°. The contours are at 2σ intervals. The peaks representing fivefold positions are delineated by the black pentagon.