Characterisation of an improved two-dimensional p22121 crystal from bovine rhodopsin.

Dialysis of rhodopsin isolated from bovine rod outer segments resulted in the formation of a new two-dimensional crystal form suitable for electron crystallography. The crystals obtained were tubular or single layers and showed p22121 symmetry (a=60.6(+/-0.8) A, b=86.3(+/-1.6) A). For the first time the size and order of the crystals allowed us to take electron diffraction patterns showing spots to a resolution of about 3.5 A. Images were recorded at liquid nitrogen temperature using a high voltage field emission electron microscope. Out of a large number of images 20 crystalline areas were selected and processed with the MRC image processing software. A projection structure of bovine rhodopsin to 5 A resolution was calculated using amplitudes and phases extracted from these images. The achieved resolution exceeds the resolution of all previously obtained structures of frog, bovine and squid rhodopsin crystals. In this map small differences are observed compared to the previous maps. Helix 5 seems to be even more highly tilted and between the arc-shaped feature and helix 5 a peak is present suggesting that helix 3 is prolonging this feature towards helix 5. These observations are in agreement with the latest model for the three-dimensional arrangement of rhodopsin. The resolution achieved as well as the availability of electron diffraction data suggest that there is a good possibility to collect data from tilted crystals and calculate an improved three-dimensional structure of rhodopsin.

[1]  N. Unwin.  Nicotinic acetylcholine receptor at 9 A resolution. , 1993, Journal of molecular biology.

[2]  J. Baldwin.  The probable arrangement of the helices in G protein‐coupled receptors. , 1993, The EMBO journal.

[3]  H. Khorana,et al.  Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187. , 1990, The Journal of biological chemistry.

[4]  J. Baldwin,et al.  An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. , 1997, Journal of molecular biology.

[5]  H. Shichi,et al.  Biochemistry of visual pigments. I. Purification and properties of bovine rhodopsin. , 1969, The Journal of biological chemistry.

[6]  P. Hargrave.  The amino-terminal tryptic peptide of bovine rhodopsin. A glycopeptide containing two sites of oligosaccharide attachment. , 1977, Biochimica et biophysica acta.

[7]  W. J. Grip,et al.  Purification of bovine rhodopsin over concanavalin A--sepharose. , 1982 .

[8]  B. Gowen,et al.  Projection structure of an invertebrate rhodopsin. , 1996, Journal of structural biology.

[9]  P. Hargrave,et al.  Rhodopsin and phototransduction. , 1992, International review of cytology.

[10]  C. Strader,et al.  The family of G‐protein‐coupled receptors , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[11]  E. E. Fesenko,et al.  Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment , 1985, Nature.

[12]  R. Henderson,et al.  A high sensitivity imaging detector for electron microscopy , 1995 .

[13]  Gebhard F. X. Schertler,et al.  Projection structure of rhodopsin , 1993, Nature.

[14]  Nicotinic Acetylcholine Receptor , 1986, NATO ASI Series.

[15]  J. Lepault,et al.  Structure of purple membrane from halobacterium halobium: recording, measurement and evaluation of electron micrographs at 3.5 Å resolution , 1986 .

[16]  N. Unwin.  Acetylcholine receptor channel imaged in the open state , 1995, Nature.

[17]  Yoshinori Fujiyoshi,et al.  Atomic model of plant light-harvesting complex by electron crystallography , 1994, Nature.

[18]  H. G. Khorana,et al.  Palmitoylation of bovine opsin and its cysteine mutants in COS cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[19]  H. Khorana,et al.  Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  J. Wess,et al.  Muscarinic acetylcholine receptors: structural basis of ligand binding and G protein coupling. , 1995, Life sciences.

[21]  G. Schertler,et al.  Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy. , 1995, Biophysical journal.

[22]  R. Henderson,et al.  Three-dimensional structure determination by electron microscopy of two-dimensional crystals. , 1982, Progress in biophysics and molecular biology.

[23]  P. Hargrave,et al.  Projection structure of frog rhodopsin in two crystal forms. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Bullough,et al.  High-resolution spot-scan electron microscopy of microcrystals of an alpha-helical coiled-coil protein. , 1990, Journal of molecular biology.

[25]  R Henderson,et al.  Analysis of electron microscope images and electron diffraction patterns of thin crystals of phi 29 connectors in ice. , 1994, Journal of molecular biology.

[26]  R Henderson,et al.  Three-dimensional structure of halorhodopsin at 7 A resolution. , 1995, Journal of molecular biology.

[27]  R A Crowther,et al.  MRC image processing programs. , 1996, Journal of structural biology.

[28]  Gebhard F. X. Schertler,et al.  Arrangement of rhodopsin transmembrane α-helices , 1997, Nature.

[29]  Kenneth H. Downing,et al.  Structure of the αβ tubulin dimer by electron crystallography , 1998, Nature.

[30]  Richard Henderson,et al.  Use of spot-scan procedure for recording low-dose micrographs of beam-sensitive specimens , 1987 .

[31]  Kenneth A Jacobson,et al.  Molecular architecture of G protein‐coupled receptors , 1996, Drug development research.

[32]  R. Henderson,et al.  Projection structure of halorhodopsin from Halobacterium halobium at 6 A resolution obtained by electron cryo-microscopy. , 1993, Journal of molecular biology.

[33]  R. Henderson,et al.  Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. , 1990, Journal of molecular biology.

[34]  F. Zemlin.  A practical procedure for alignment of a high resolution electron microscope , 1979 .

[35]  J. Baldwin,et al.  Structure and function of receptors coupled to G proteins. , 1994, Current opinion in cell biology.

[36]  R Henderson,et al.  Electron-crystallographic refinement of the structure of bacteriorhodopsin. , 1996, Journal of molecular biology.