Cryo-EM Analysis of Photosynthetic Complexes: From Purification to Data Collection

Oxygenic photosynthesis, the process that converts solar energy into chemical energy, begins with light capture by pigment-protein complexes in thylakoid membranes. Cryo-electron microscopy (Cryo-EM) can determine the structures of these complexes at near-atomic resolution, thereby unveiling the molecular basis of light harvesting, excitation energy transfer, and dissipation, and their regulation. Here, we present a workflow for studying plant photosynthetic complexes using Cryo-EM, from isolation and purification of pigment-protein complexes to sample preparation and data acquisition. Using plant Photosystem I as a case study, the protocol provides guidance on grid preparation, vitrification, and the collection of high-quality datasets. It further underscores how purification and sample-preparation strategies can be adapted to different plant sources and photosystem populations, while preserving the stability and integrity of these labile membrane assemblies. Coupled to Cryo-EM parameters tailored to the specific biophysical properties of distinct photosynthetic complexes, this workflow provides a robust and reproducible route from leaf tissue to near-atomic-resolution structures.