2-DIMENSIONAL CRYSTALLIZATION AND CRYOELECTRON MICROSCOPY OF PHOTOSYSTEM-II

Two-dimensional (2D) crystals of photosystem II (PSII) were obtained f rom n-heptyl-beta-D-thioglucoside-solubilized monomers of spinach PSII complex by conventional detergent dialysis. The 2D crystals were eith er large cylindrical vesicles (1 to 2 mu m by 4 to 6 mu m as flattened vesicles) or large monolayer sheets (ca. 1 mu m x 1 mu m), both suita ble for cryo-electron microscopy Images of unstained crystals embedded in ice were recorded using low-dose microscopy and analyzed by digita l image processing. Both types of crystals had the same unit cell size and the same packing arrangement of PSII particles. The plane group w as rho 22(1)2(1) and the unit cell was rectangular with dimensions of 16.7 nm x 15.3 nm containing four monomers (two face-up and two face-d own). SDS-PAGE and immunoblot analyses of the 2D crystal indicated tha t the constituent subunits of particles in the 2D crystal were CP47, D 1, D2, cytochrome b-559 and psbI protein. A projection map at 20 Angst rom resolution revealed that each monomer has an asymmetrical shape wi th a length of 8.1 nm and a maximal width of 7.5 nm consisting of four areas of density. Two high-density areas with similar sizes were loca ted close to each other to form a roughly rectangular core of 4.0 nm x 6.5 nm. From its size similarity to the size of the L/M heterodimer o f the bacterial reaction center, this high density core area was tenta tively assigned to the D1/D2 heterodimer. The remaining large and smal l areas were also tentatively assigned to CP47 and cytochrome b-559 pl us psbI protein, respectively. (C) 1996 Academic Press Limited