Three-dimensional structure of bovine rhodopsin determined by electron microscopy

G-protein coupled receptors mediate signal transduction through the cell membrane. The visual pigment rhodopsin is the best characterised receptor of this large family of proteins. At present, the three-dimensional structure of rhodopsin has been determined to a resolution of 7.5 Å in the plane and 16.5 Å perpendicular to it. Images of two- dimensional crystals of bovine rhodopsin have been analysed and a projection map to 5 Å has been calculated (Krebs et al., submitted to Journal of Molecular Biology). Preliminary calculations of a three-dimensional data set show good compatibility of the data to a resolution of about 9 Å in the membrane plane. In this project the aim is to improve the completeness, quality and resolution of the three-dimensional data so that for the first time the seven transmembrane helices can be resolved properly. Two major steps are planned: a) Additional images of tilted specimens will be taken at liquid nitrogen temperature using a high voltage (200kV) field emission electron microscope. Data extracted of these images will be combined with the already existing data leading to a complete, preliminary three-dimensional data set of the structure of rhodopsin. b) "Cyclic refinement" using this preliminary data set as reference will be tried. The quality of our current data indicates that with a complete three-dimensional data set it should be possible to improve the resolution using "cyclic refinement". These additional steps will enable us to determine the exact location of all seven transmembrane helices and to build a more detailed model of rhodopsin which will then provide a better insight into signal transduction in general.