Biochemical and cryo-EM studies of the pyruvate dehydrogenase complex from native cell extracts.

Thiamine pyrophosphate (TPP)- dependent multi-enzyme dehydrogenase complexes are central regulators of metabolism that catalyze the oxidative decarboxylation of α-ketoacids, and link central pathways such as glycolysis and the Krebs cycle. These large, multimeric assemblies span pyruvate, oxoglutarate, and branched-chain α-ketoacid dehydrogenase complexes and coordinate multiple catalytic activities through a sophisticated macromolecular organization and substrate channeling via flexible lipoyl arms. Pyruvate dehydrogenase complex (PDHc) plays a key regulatory role in processes such as carbon flux control, energy production, and redox balance, while also participating in non-canonical functions, including histone acetylation and regulation of gene expression in the nucleus. Despite extensive research over many decades, we still lack a clear understanding of how these complexes are structurally organized and dynamically coordinated, largely due to their size, heterogeneity, and flexibility. Recent advances in cryo-electron microscopy and near-native biochemical and structural approaches are beginning to uncover their higher-order architecture. Minimal purification strategies, combined with suitable model organism selection, provide promising approaches to preserve native interactions and to further mechanistic understanding of these fundamental metabolons. Here, we provide a detailed protocol from model organism growth to cryo-EM Coulomb potential map generation, focusing on retrieving native, endogenous PDHc for structural and functional analysis. We offer practical examples while highlighting challenges in experimental procedures and data analysis. Although the protocols were optimized for the model organism described here, the workflow was successfully applied to other systems, including yeast cells and human cell lines, with parts also adapted for plant cells.