Fast-forward pipeline for cryo-EM structural analysis of native plant protein complexes

*This is a poster for British Society of Plant Pathology (BSPP) conference - Plant Pathology 2024 (PPATH2024), Oxford Fast-forward pipeline for cryo-EM structural analysis of native plant protein complexes Selvaraj Muniyandi (1), Hsuan Pai (1), Jogi Madhuprakash (1), AmirAli Toghani (1), Michael Webster (2), Sophien Kamoun (1), and Mauricio Contreras (1) (1) The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, United Kingdom (2) John Innes Centre, Norwich Research Park, Norwich, United Kingdom Nucleotide-binding domain leucine-rich repeat (NLR) proteins play a vital role in plant innate immunity, forming oligomerized complexes called resistosomes upon activation. NLRs can operate individually or in higher-order configurations, such as pairs or networks, displaying functional connections between specialized sensor and helper proteins. Previous studies have utilized the hypersensitive response (HR) assay to screen functional NLRs and their target effectors, alongside blue native polyacrylamide gel electrophoresis (BN-PAGE) to visualize the oligomeric status of resistosomes upon effector perception. However, understanding the biological process of NLR activation requires bridging these macroscopic readouts with molecular and biochemical mechanisms, which remains challenging due to the difficulty in resolving NLR protein complex structures in their native state. Here, we showcase two fast-forward methods, immunoprecipitation – electron microscopy (IP-EM) and BN-PAGE EM (BN-EM), involving small-scale protein purification but capable of deciphering structures of large protein complexes using cryo-EM. Through IP-EM, we showed that the helper NLR required for cell death (NRC) exists as an inactive homodimer, transitioning to a hexameric state upon effector perception triggered by its cognate sensor NLR. These findings highlight the diversity of forms and modes of action in resistosome activation. Additionally, we determined the structure of the RuBisCO enzyme as a test case for cryo-EM of protein complexes electro-eluted from BN-PAGE. The BN-EM pipeline enabled the determination of the Nicotiana benthamiana and rice hexadecameric RuBisCO structures at 3.4Å resolution. Together, these methods represent versatile tools for fast-forward elucidation of a broad range of plant protein complexes.