Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed. In this review, we give an overview of both commonly used and recently developed biochemical methodologies for covalent stabilization of macromolecular complexes enabling structural investigation via crystallography, nuclear magnetic resonance, and cryo-electron microscopy. We divided the strategies into nonenzymatic- and enzymatic-driven cross-linking and further categorized them in either naturally occurring or engineered covalent linkage. This review offers a compilation of recent advances in diverse scientific fields where the structural characterization of macromolecular complexes was achieved by the aid of intermolecular covalent linkage.
