Nature © Macmillan Publishers Ltd 1997 Arrangement of rhodopsin transmembrane -helices Vinzenz M. Unger*†, Paul A. Hargrave‡, Joyce M. Baldwin* & Gebhard F. X. Schertler* * MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK ‡ Department of Ophthalmology, and Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA † Present address: The Scripps Research Institute, Department of Cell Biology- MB28, 10666 North Torrey Pines Road, La Jolla, California 92037, USA. ......................................................................................................................... Rhodopsins 1 , the photoreceptors in rod cells, are G-protein- coupled receptors with seven hydrophobic segments containing characteristic conserved sequence patterns that define a large family 2,3 . Members of the family are expected to share a conserved transmembrane structure. Direct evidence for the arrangement of seven -helices was obtained from a 9A ˚ projection map of bovine rhodopsin 4 . Structural constraints inferred from a comparison of G-protein-coupled receptor sequences were used to assign the seven hydrophobic stretches in the sequence to features in the projection map 5 . A low-resolution three-dimensional structure of bovine rhodopsin 6 and two projection structures of frog rhodopsin 7 confirmed the position of the three least tilted helices, 4, 6 and 7. A more elongated peak of density for helix 5 indicated that it is tilted or bent 6,7 , but helices 1, 2 and 3 were not resolved. Here we have used electron micrographs of frozen-hydrated two- dimensional frog rhodopsin crystals to determine the structure of frog rhodopsin. Seven rods of density in the map are used to estimate tilt angles for the seven helices. Density visible on the extracellular side of the membrane suggests a folded domain. Density extends from helix 6 on the intracellular side, and a short connection between helices 1 and 2, and possibly a part of the carboxy terminus, are visible. The extraction of frog rod cell membranes with Tween 80 resulted in the formation of two-dimensional crystals of rhodopsin with P2 symmetry 7 . The crystals were better ordered than two-dimensional crystals obtained previously by reconstitution of detergent-purified bovine rhodopsin in synthetic lipids 4 . From several hundred images of tilted specimens, 60 crystalline areas were selected by optical diffraction, and amplitudes and phases were extracted from the electron micrographs 4,6,8 . Because the elongated shape and small size of the coherent diffracting areas precluded the recording of electron diffraction patterns, image-derived amplitudes were used together with the phase values obtained from sampling the lattice lines at 0.005 A ˚ -1 . Three lattice lines are shown in Fig. 1. The distribution of the data is shown in Fig. 2, together with the point-spread function 6 of the fitted data set. The three-dimensional map had an effective resolution of 7.5 A ˚ in the membrane plane and 16.5 A ˚ normal to it. A single rhodopsin molecule in this map has planar dimensions of 28  39 ˚ A. The molecule appears 64 A ˚ high at the chosen contour level of Fig. 3 but, because of the low vertical resolution, this is only an approximation. In contoured cross-sections taken parallel to the membrane plane the clearest features are close to the middle of the membrane (Fig. 4). The central sections of the seven transmem- brane helices are marked in Fig. 3 with lines starting at section +12 A ˚ and ending at section -8A ˚ . This part of the map is contained within the hydrophobic core of the bilayer. The density peaks representing the seven helices have been interpreted according to the previous assignment of sequence segments 5 to the projection map of bovine rhodopsin 4 . This assign- ment is not confirmed directly by the current map as the amino and carboxy termini and the connections between the helices are not clearly identified, but all features seen in the map are consistent with this assignment. The map confirms the positions of the three least- tilted helices, 4, 6 and 7, and also shows features that can be assigned to tilted helices 1, 2, 3 and 5, which were not resolved in the previous maps. The centres of the peaks on section +12 A ˚ and section -8A ˚ (Fig. 4) were used to calculate tilt angles for seven transmembrane helices. These tilt angles, which ignore possible helix curvature or kinks but give an indication of the tilt direction for each helix, are shown in Table 1. The density feature assigned to helix 1 has an approximate overall tilt angle of 28 deg. The extracellular end is highly exposed to the lipid, which is consistent with this part of the sequence being variable in the opsin gene family (see ref. 9). Beyond section +12 A ˚ , the density in the region of helix 1 separates into two letters to nature NATURE | VOL 389 | 11 SEPTEMBER 1997 203 Table 1 Estimate of the axes of the seven helices Orientation Position Helix v (deg) f (deg) x 0 (A ˚ ) y 0 (A ˚ ) ............................................................................................................................................................................. 1 28.4 141.0 -2.16 7.52 2 27.2 82.2 -6.24 15.08 3 29.6 50.7 -1.92 23.92 4 3.8 116.6 -7.04 30.08 5 22.7 -11.0 5.08 34.56 6 7.4 -90.0 10.40 23.16 7 13.4 -165.4 6.36 15.32 ............................................................................................................................................................................. The axes are determined from the coordinates of the observed peaks on Z-sections -8A ˚ and +12 A ˚ (Fig. 4). The crystallographic b axis is horizontal in Fig. 4, and the c axis is perpendicular to the plane of the membrane, with +c (+Z) pointing towards the intracellular side. Because the crystallographic axes are not orthogonal, coordinates are referred to orthogonal axes X and Y , where Y is parallel to b and X is perpendicular to b and c (X is very nearly parallel to a). v is the angle between the helix axis and the direction of the +Z axis; f is the angle around the +Z axis measured from the direction of the +X axis; a positive f angle indicates a right-handed rotation about +Z, (positive f towards +Y , negative f towards -Y); x0, y0 are the coordinates (A ˚ ) along the X, Yaxes at which the helix axis intersects section z ¼ 0. Figure 1 Three typical lattice lines. The top panel for each lattice line shows the change of phase along the z*-axis. The bottom panel shows the changing amplitude (in arbitrary units) along the z*-axis. The final data set obtained from 60 tilted images contains a total of 3,378 raw measure- ments above background. These were used for fitting of the lattice lines in space group P2 (a ¼ 32 ˚ A, b ¼ 83 ˚ A, g ¼ 91 deg). Error bars show the fitting error at the sampling points, and are spaced at 0.005 A ˚ -1 along the z* axis, normal to the crystal plane.