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.