Extended Data Fig. 1: Characterizations of the Arabidopsis PhyA preparations and the workflow used for processing its cryo-EM images.

a, SDS-PAGE analysis of the recombinant full-length PhyA. Gels were either stained for protein with Coomassie blue (left) or assayed for bound PΦB by zinc-induced fluorescence (right). MM, molecular mass standards. Samples were indistinguishable to those described by Burgie et al.⁶ b, UV-vis absorbance spectra of PhyA. The spectra were collected from dark-adapted samples (Pr) or after saturating irradiation with 630-nm red light (RL, mostly Pfr). Absorption maxima were determined from the difference spectrum shown at 70% amplitude. The SCR at 664/723 nm is indicated in parenthesis. Spectra were the average of three technical replicates. c, Workflow used for data processing of the cryo-EM images of PhyA. In the first refined overall map at 3.5-Å resolution based on 421,969 particles, almost all PhyA domains were well seen except for the regions encompassing the PAS1 domains, which were poorly resolved. Focused refinements, excluding the PAS1 domains and using signal subtraction for the PSM, PAS2 and HKRD regions, generated a 3.2-Å map with some ambiguity in the HKRDs. Subsequent use of separate masks for the HKRDs and the platform led to improved 3.1-Å and 3.4-Å EM maps for the platform and HKRDs, respectively, which were combined to generate the final composite map of the dimer. 3DVA of particle images down-sampled to 4.14 Å per pixel resolved one of the flexible PAS1 domains (purple) at ~15-Å resolution. d, A representative cryo-EM micrograph after motion correction is shown. In total, data from 6,195 independent micrographs were utilized for map construction. e, Selected 2D class averages showing multiple views of the particles.