Illuminating ubiquitination mechanisms: How cryo-EM has shed light on Cullin RING E3 ligase function

Abstract

The ubiquitin-proteasome system (UPS) governs protein homeostasis by orchestrating the selective degradation of regulatory and misfolded proteins through a tightly regulated series of ATP-driven ubiquitination reactions. E3 ubiquitin ligases play a central role in this process by conferring substrate specificity, yet the structural complexity and dynamic nature of these large macromolecular assemblies poses challenges for traditional structural biology techniques such as X-ray crystallography and nuclear magnetic resonance (NMR). The advent of single-particle cryo-electron microscopy (cryo-EM) has transformed our ability to study these enzymes, revealing previously inaccessible mechanistic insights into their allosteric regulation, conformational transitions, and substrate recognition. By integrating high-resolution crystallographic data with cryo-EM's ability to resolve heterogeneous and dynamic complexes, researchers have uncovered fundamental principles governing E3 ligase activity. This review explores how cryo-EM has reshaped our understanding of Ligases. We highlight key discoveries enabled by this technique, and discuss how emerging cryo-EM approaches, alongside complementary methodologies, are advancing therapeutic strategies targeting ubiquitin signaling by this family of ligases.