(A) Three segmented thylakoids from the region indicated in Figure 1B. Membranes 2–5 (M2 to M5, alternating green and magenta) are examined by membranograms. The eye symbol with arrow indicates the viewing direction for the membranograms. (B) Overlays of two luminal surface membranograms, superimposed across the stromal gap (M2-M3, M4-M5) and thylakoid lumen (M3-M4). Membranograms are pseudocolored corresponding to A. Overlapping PSII complexes in the M4-M5 overlay are indicated with white arrowheads. (C) Model representations of M4 and M5, with C2S2M2L2-type PSII-LHCII supercomplexes positioned according to the luminal densities observed in the membranograms. The spacing indicates that appressed thylakoids can accommodate these supercomplexes. (D) M4-M5 overlays using the membrane models from C, separately showing the PSII core complexes (top) and LHCII antennas (bottom). Overlapping regions are white. White arrowheads correspond to the overlapping complexes in B. (E) Blue bar: the percentage of membrane surface area occupied by luminal EM density (15.0 ± 1.2%, N = 19 membranes) in membranograms (e.g., panel B). Red and red/yellow striped bars: the percentage of surface area in membrane models (e.g., panel C) occupied by C2-type PSII core complexes (14.6 ± 4.1%) and C2S2-type (29.1 ± 3.7%), C2S2M2-type (40.7 ± 5.2%), and C2S2M2L2-type (47.3 ± 6.0%) PSII-LHCII supercomplexes (N = 9 membranes). (F) Blue bars: the percentage of luminal EM density in membranograms that overlaps between adjacent appressed membranes spanning the stromal gap (7.2 ± 1.1%, N = 11 overlays) and thylakoid lumen (4.0 ± 0.5%, N = 6 overlays). See Figure 4—figure supplement 1 for how the EM density was thresholded to calculate surface area and overlap. Red and yellow bars: using membrane models with C2S2M2L2-type PSII-LHCII supercomplexes, the percentage of PSII and LHCII surface area that overlaps between adjacent appressed membranes spanning the stromal gap (N = 4 overlays). The experimental measurements (exp.; PSII: 8.0 ± 1.9%, LHCII: 12.9 ± 2.5%) and simulations of complexes randomly positioned within the membrane (random; PSII: 8.3 ± 1.4%, LHCII: 13.8 ± 1.6%, N = 100 simulations per overlay) were not significantly different (p>0.05 from t-tests with Welch’s correction for unequal variances: p=0.512 for PSII, p=0.762 for LHCII). See Figure 4—figure supplement 2 and Materials and methods for how the random simulations were performed. Error bars denote standard deviation.