Extended Data Fig. 1: Overview of FIB lamella sample preparation and flowchart of cryo-EM study.
From: In situ structure of the red algal phycobilisome–PSII–PSI–LHC megacomplex
a, A representative EM image of P. purpureum lamella at low magnification. 46 tilt series images were collected with similar results. b, A representative EM image of P. purpureum lamella (selected from 46) at media magnification. 46 similar tilt series were collected . c, A flowchart of the cryo-EM analysis. 23,637 sub-tomogram particles were picked out from tomograms reconstructed from 46 tilt series in emClarity. Alignment and averaging of subtomograms yielded an 8 Å structure. 3D classification showed two distinct conformations, the single and double PBS-PSII–PSI-LHC megacomplexes. The tilt images corresponding to the all subtomograms were extracted and subjected to local refinement in RELION with a soft-edge mask of PBS, yielding a 6.5 Å structure of PBS. This structure was then used as a high-resolution reference to pick out the potential particles(762,000) in high-dose image data (2,245) by method isSPA. After further particle filtering, 3D classifications and refinements, we obtained high resolution structures of single and double PBS-PSII-PSI-LHC megacomplexes from 215,000 and 87,000 particles, respectively.
