a, Sucrose gradient of solubilized grana membranes. The membrane preparations were first washed with 1 mM (left) or 5 mM (right) EDTA before being solubilized by α-DDM for further purification through sucrose-gradient ultracentrifugation. The content of each band is indicated based on the absorption spectrum and SDS–PAGE results, and by comparing to previously published data. The B9 fraction obtained from the grana membrane washed with 1 mM EDTA was used for cryo-EM. Note that the grana membranes washed with 1 mM EDTA yielded less B5, B6 and B7 than the sample treated with 5 mM EDTA. b, SDS–PAGE analysis of the sucrose gradient fractions. The protein composition of each Coomassie band was indicated based on the mass spectrometry and proteomics data analysis. For gel source data, see Supplementary Fig. 1. c, Room-temperature absorption spectrum of B9 sample used for cryo-EM. Its spectrum (B9 1 mM) is compared to those of B7 (dimeric PSII core without LHCII attached; B7 5 mM) and B9 samples (B9 5 mM) fractionated from grana washed with 5 mM EDTA. Note that B9 from grana membranes washed with 1 mM EDTA showed higher peaks at 470 and 650 nm, indicating that this fraction contains higher Chl b content (from LHCIIs) than the other two. The spectra are normalized to the maximum in the red region. d, Fluorescence emission spectra of B9 sample measured at room temperature. The maximum emissions were at 681 nm (upon excitation of Chl a at 436 nm), 680 nm (upon excitation of Chl b at 473 nm) and 681 nm (upon excitation of carotenoids at 500 nm). Overlapping of these three spectra suggests that nearly all pigments in the B9 sample are well coupled and no free pigments are present. e, Pigment content analysis of B9 sample by HPLC. Based on the characteristic absorption spectrum of each peak fraction, the six major pigment peaks separated from the B9 sample are identified as neoxanthin (Neo), violaxanthin (Vio), lutein (Lut), Chl b, Chl a and β-carotene (β-car).