2d crystallisation and electron diffraction pattern

Isolation and crystallisation of the protein was carried out in dim red light. Rhodopsin, purified from bovine rod outer segments, was solubilised in lauryldimethylamine oxide and dialysed in 20 mM HEPES, 2 mM dithiothreitol, pH 7.5 at 18° C for 11 days. The obtained suspension was purified on a sucrose gradient and crystals were found in the fraction with a density of 1.166 g/ml (Fig.1a). The concentration and state of rhodopsin was determined spectroscopically (10).
Electron diffraction patterns of exceptionally large single layers (Fig. 1b) were taken at 120 kV using a Phillips CM12 electron microscope and a large area, cooled CCD detector (11). All work was carried out at liquid nitrogen temperature.