Part 4 Results (cont.)

Electron Microscopy - To investigate further the nature of the LHCI-PSI complex in F3, the contents of this fraction were viewed by EM after negative staining and the images processed by single particle analysis. Fig. 4a shows an averaged top view obtained by processing 8950 particles, which has maximum dimensions of 220 . 180 Å. To interpret this projection map the EM structure of the PSI complex of Chlamydomonas, free of LHCI protein, was investigated. To this end, images of fraction F2 were recorded and subjected to single particle analysis. After multistatistical analysis of the data set it was clear that the F2 fraction contained two subpopulations based on their surface area and differences in domain organization. The subpopulation with a two-domain character were assigned to the PSII dimeric core present in F2, which is known to have dimensions of 220 . 150 Å in negative stain (not shown) (30, 31), while the other major population contains particles having a single domain and dimensions of 150 . 100 Å, indicative of a PSI core monomer. Fig. 4c shows the average top view of the Chlamydomonas PSI monomer obtained by processing 680 particles. Of note is that neither in fractions F2 nor F3 did we observe larger particles that would suggest oligomerization of the PSI monomer and, in particular, the existence of a trimer similar to that found for PSI in cyanobacteria (2). The LHCIPSI supercomplex projection shown in Fig. 4a has strong density features along one side. We assign these features to the Lhca proteins present. To focus on and improve the quality of these features, we overlaid the PSI monomer projection onto the projection of the supercomplex and used this as a mask to conduct further rounds of image analyses utilizing the whole supercomplex data set. The resulting image is shown in Fig. 4b. The overlaying of the PSI monomer projection onto that of the supercomplex was aided by reference to the x-ray structure of PSI derived by Jordan et al. (2). In the x-ray map the PsaL subunit, with its three transmembrane helices, forms a distinctive bulge at one end of the monomer. We therefore assumed that a similar feature in the projection maps of the supercomplex and monomer (starred in Fig. 4, a and c) are due to PsaL and used this for an approximate alignment of the maps (Fig. 4d). This comparison further suggests that the top views of the supercomplex and monomer (shown in Fig. 4, a and c) are being viewed from the stromal side since the feature assigned to PsaL lies left of center in the stromal top view of the x-ray-derived PSI asymmetric monomer (see red ring; Fig. 4d). As our native EM data set contained images of the LHCI-PSI supercomplex in different orientations we have obtained a range of class averages and calculated a low resolution, threedimensional model. Fig. 5 shows typical class averages taken from the total pool of 76 class averages and shows a range of orientations. All 76 class averages were used to construct the three-dimensional model presented in Fig. 5c as surface-rendered views and at the same orientation as the class averages given in Fig. 5a. The calculated projections derived from the three-dimensional model are shown in Fig. 5b and, according to Fourier shell correlation analysis, have a resolution of about 30 Å.