Two-dimensional Crystallization and Cryo-electron Microscopy of Photosystem II

Two-dimensional (2D) crystals of photosystem II (PSII) were obtained from n-heptyl-β-D-thioglucoside-solubilized monomers of spinach PSII complex by conventional detergent dialysis. The 2D crystals were either large cylindrical vesicles (1 to 2 μm by 4 to 6 μm as flattened vesicles) or large monolayer sheets (ca. 1 μm × 1 μm), both suitable for cryo-electron microscopy. Images of unstained crystals embedded in ice were recorded using low-dose microscopy and analyzed by digital image processing. Both types of crystals had the same unit cell size and the same packing arrangement of PSII particles. The plane group wasp22121and the unit cell was rectangular with dimensions of 16.7 nm × 15.3 nm containing four monomers (two face-up and two face-down). SDS-PAGE and immunoblot analyses of the 2D crystal indicated that the constituent subunits of particles in the 2D crystal were CP47, D1, D2, cytochromeb-559 andpsbI protein. A projection map at 20 Å resolution revealed that each monomer has an asymmetrical shape with 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 located close to each other to form a roughly rectangular core of 4.0 nm × 6.5 nm. From its size similarity to the size of the L/M heterodimer of the bacterial reaction center, this high density core area was tentatively assigned to the D1/D2 heterodimer. The remaining large and small areas were also tentatively assigned to CP47 and cytochromeb-559 pluspsbI protein, respectively.