The 2D crystallization method using a liquid-liquid interface has been applied to the Rhodococcus 20S proteasome, Two types of ordered arrays were obtained, both large enough for high-resolution analysis. The first one is a hexagonal close-packed array, whereas the second one has fourfold symmetry. By image analysis based on a real space correlation averaging technique, the close-packed array was found to be hexagonally packed hut the molecules had complete rotational freedom. The fourfold array is, however, a true crystal, with p4 symmetry. Lattice constants are a = b = 20.0 nm and the unit cell of this crystal contains two proteasomes. The diffraction pattern computed from the original picture shows the spots up to (4.5) that correspond to 3.1 nm resolution. After applying an unbending procedure, the diffraction pattern shows spots extending to 1.8 nm resolution. (C) 1996 Academic Press, Inc.