Part 1

The dimeric PSII supercomplex used for this study was isolated from spinach chloroplast thylakoid membranes. Each monomer, within the dimer, contains the reaction centre proteins D1 and D2 and the inner antenna proteins, CP43 and CP47 (1). It also contains one trimer of LHCII and a single copy each of the chlorophyll a/b binding proteins, CP26 and CP29 (1). It may also contain the PsbS protein (2). Importantly, the isolated supercomplex retains the three major lumenal bound extrinsic proteins of the OEC; 33 kDa, 23 kDa and 17 kDa proteins (see Fig. 1a). The 33 kDa protein stabilizes a cluster of four Mn atoms, which forms the catalytic site for the water oxidation reaction. The 23 and 17 kDa proteins seem to play a role in maintaining optimal levels of Ca2+ and Cl- for the water oxidation reaction (3). The light induced electron transfer processes, which drive the water splitting reaction, involve redox active cofactors associated with the D1 and D2 proteins. These reactions generate a very high oxidising potential in the region of 1Volt that is initially located on the special form of chlorophyll a known as P680. The Mn cluster donates electrons to P680+ via a redox active tyrosine located at position 161 on the D1 protein (3). Four oxidising equivalents are stored in this way and are used to generate one dioxygen from two water molecules. Since the isolated PSII supercomplex contains all subunits of the OEC, it is highly active in light driven oxygen evolution (2). Figs. 1b and c show a typical focal pair of electron micrographs of the isolated PSII supercomplex preparation in vitreous ice without staining. The particles visualised are randomly orientated. The near to focus micrographs (Fig. 1b, 1.35 µm defocus) were used for analyses, whereas the more underfocused images (Fig. 1c, 7.2 µm defocus) were used to facilitate particle identification. Using a data set of 15,650 particles, 750 class averages were identified initially of which 439 representing 5,300 particles were used for the 3D reconstruction of the supercomplex, exploiting a D2 point group symmetry established at the early stages of the analysis. Fig.1d shows six characteristic class average views of the supercomplex. The relative orientations were determined for each class average and were used to construct the 3D map shown in Fig. 1f and corresponding reprojections in Fig. 1e. It can be seen that the supercomplexes aggregate to form dimers of dimers via interactions at the stromal surface and this gives rise to the D2 point group assignment. The protrusions clearly observed on the lumenal surfaces are due to the extrinsic proteins of the OEC (4). The 3D model has been corrected for the Phase Contrast Transfer Function (CTF). The resolution of the structure is 24 Å as determined by Fourier Shell Correlation (FSC) analysis (5,6).

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