Figure 1. Aquaporin-0 (AQP0) forms two-dimensional (2D) crystals with all tested sphingomyelin/cholesterol mixtures.

(A–E) AQP0 purified from sheep lenses was reconstituted with pure sphingomyelin (A), sphingomyelin/cholesterol mixtures at molar ratios of 2:1 (B), 1:2 (C), and 1:4 (D), as well as pure cholesterol (E). AQP0 was reconstituted under all conditions and formed diffracting 2D crystals. The scale bars are 2 μm. (F) Projection map of AQP0 reconstituted with pure cholesterol at 3.2 Å resolution. The 2D crystals show p422 symmetry and have the typical lattice constants for AQP0 crystals of a=b=65.5 Å, and γ=90°. The panel shows two-by-two unit cells. See also Figure 1—figure supplements 1 and 2 and Tables 1 and 2.

Figure 1—figure supplement 1. Chemical structures of raft lipids.

(A) Cholesterol consists of a planar four-aromatic ring structure (red) with a short isooctyl alkyl chain (black) and a small 3-β-hydroxyl head group (blue). The side view shows the asymmetric distribution of the aliphatic groups linked to the ring system and the resulting smooth (α) and rough (β) faces of the cholesterol molecule. (B) Sphingomyelin consists of a sphingosine amino alcohol (red) with a fatty acid (black) and phosphocholine head group (blue).

Figure 1—figure supplement 2. Diffraction of an image of an aquaporin-0 (AQP0) two-dimensional (2D) crystal grown with pure cholesterol.

(A) Power spectrum of a raw image of an AQP0 2D crystal in a pure cholesterol membrane. The scale bar represents (10 Å)^–1. (B) Intensity quotient (IQ) plot after computational lattice unbending. After lattice unbending of the image in 2dx (Gipson et al., 2007b), the IQ plot showed reflections with IQ values of 3 (corresponding to a peak-to-background ratio of 2.3) to a resolution better than 4 Å.