Part 3 Materials and Methods (cont.)

Electron Microscopy and Image Processing-Preparations were negatively stained with 2% uranyl acetate on glow-discharged carbon-evaporated grids and imaged using a Philips CM 100 electron microscope at 80 kV. The magnification was calibrated at 51,500x. Twenty electron micrographs were taken for each preparation and subsequently calculated to have the first minima of their contrast transfer functions to be in the range of 17-23 Å. Electron micrographs were digitized using a Leafscan 45 densitometer set at a step size of 10 um. Single particle data sets of ~3000 (CP43'-PSI supercomplex) and 4200 (PSI trimer) were obtained by interactively selecting all possible particles from the micrographs. All subsequent processing was performed within the IMAGIC-5 software environment (22, 23). The single particle images were coarsened by a factor of 2, resulting in a sampling frequency of 3.88 Å/pixel on the specimen scale. Reference-free alignment coupled with multi-variate statistical analysis was used to classify each data set to identify initial class averages. These data were then used for iterative refinement, resulting in the improved class averages. The relative orientations of the improved class averages were determined bythe angular reconstitution technique (24), allowing for an initial threedimensional reconstruction to be obtained by exact-back projection (25). Reprojections were taken from this initial three-dimensional map to further refine the class averages and identify any atypical views present within the data set. The data converged after several rounds of iterative refinement in this manner, whereby roughly 40% of the class averages were discarded after assessment through cross-correlation functions. The resolution of the final three-dimensional map was determined by Fourier shell correlation (FSC) between two independent three-dimensional reconstructions (26) compensated for the C3 symmetry used (27).

Molecular Modeling-Coordinate data sets were obtained from the Research Collaboratory Structural Bioinformatics Data Bank (www. rcsb.org) for the entry codes 1C51 (PSI 4 Å structure (28)) and 1FE1 (PSII 3.8 Å structure (29)). These structural models were visualized using the program Swiss-Protein Data Bank viewer (Glaxo-Wellcome Experimental Research (30)) and modeled into the calculated threedimensional map using the "O"-modeling software package (31).