UNCORRECTED PROOF Structure of Bovine Rhodopsin in a Trigonal Crystal Form Jade Li 1 *, Patricia C. Edwards 1 , Manfred Burghammer 2 , Claudio Villa 1 and Gebhard F. X. Schertler 1 * 1 Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, UK 2 European Synchrotron Radiation Facility, BP 220 8043 Grenoble, France We have determined the structure of bovine rhodopsin at 2.65 A ˚ resolution using untwinned native crystals in the space group P3 1 , by molecular replacement from the 2.8 A ˚ model (1F88) solved in space group P4 1 . The new structure reveals mechanistically important details unresolved previously, which are considered in the membrane context by docking the structure into a cryo-EM map of 2D crystals. Kinks in the transmembrane helices facilitate inter-helical polar interactions. Ordered water molecules extend the hydrogen bonding networks, linking Trp265 in the retinal binding pocket to the NPxxY motif near the cytoplasmic boundary, and the Glu113 counterion for the protonated Schiff base to the extracellular surface. Glu113 forms a complex with a water molecule hydrogen bonded between its main chain and side- chain oxygen atoms. This can be expected to stabilise the salt bridge with the protonated Schiff base linking the 11-cis-retinal to Lys296. The cytoplasmic ends of helices H5 and H6 have been extended by one turn. The G-protein interaction sites mapped to the cytoplasmic ends of H5 and H6 and a spiral extension of H5 are elevated above the bilayer. There is a surface cavity next to the conserved Glu134-Arg135 ion pair. The cytoplasmic loops have the highest temperature factors in the structure indicative of their flexibility when not interacting with G-protein or regulatory proteins. An ordered detergent molecule is seen wrapped around the kink in H6, stabilising the structure around the potential hinge in H6. These findings provide further explanation for the stability of the dark state structure. They support a mechanism for the activation, initiated by photo-isomerisation of the chromophore to its all-trans form, that involves pivoting movements of kinked helices, which, while maintaining hydro- phobic contacts in the membrane interior, can be coupled to amplified translation of the helix ends near the membrane surfaces. q 2004 Published by Elsevier Ltd. Keywords: G protein-coupled receptor; G protein activation; ligand binding pocket; membrane protein structure; visual pigments *Corresponding authors 0022-2836/$ - see front matter q 2004 Published by Elsevier Ltd. Abbreviations used: 2D, two-dimensional; 3D, three-dimensional; C8E4, n-octyltetraoxyethylene; C1, cytoplasmic loop connecting helices 1 and 2; C2, cytoplasmic loop connecting helices 3 and 4; C3, cytoplasmic loop connecting helices 5 and 6; cGMP, 3 0 ,5 0 -cyclic guanosine monophosphate; cryo-EM, electron cryomicroscopy; E1, extracellular loop connecting helices 2 and 3; E2, extracellular loop connecting helices 4 and 5; E3, extracellular loop connecting helices 6 and 7; EM, electron microscopy; EMTS, ethyl mercurythiosalicylate; FTIR, Fourier transform infrared spectroscopy; GPCR, G protein-coupled receptor; G-protein, heterotrimeric guanine nucleotide binding protein; Gt, transducin; Gta, transducin a-subunit; Gta 340–350, C-terminal peptide from transducin a-subunit; LDAO, N,N-dimethyldodecylamine- N-oxide; NCS, non-crystallographic symmetry; PDE, phosphodiesterase. E-mail addresses of the corresponding authors: jl@mrc-lmb.cam.ac.uk; gfx@mrc-lmb.cam.ac.uk YJMBI 56620—10/9/2004—08:47—ATHIAGARAJAN—119414—XML – pp. 1–30/TL doi:10.1016/j.jmb.2004.08.090 J. Mol. Biol. (2004) xx, 1–30 DTD 5 ARTICLE IN PRESS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126