Chemical probing provides insight into the native assembly state of a bacterial microcompartment.

Bacterial microcompartments (BMCs) are widespread in bacteria and are used for a variety of metabolic purposes, including catabolism of host metabolites. A suite of proteins self-assembles into the shell and cargo layers of BMCs. However, the native assembly state of these large complexes remains to be elucidated. Herein, chemical probes were used to observe structural features of a native BMC. While the exterior could be demarcated with fluorophores, the interior was unexpectedly permeable, suggesting that the shell layer may be more dynamic than previously thought. This allowed access to cross-linking chemical probes, which were analyzed to uncover the protein interactome. These cross-links revealed a complex multivalent network among cargo proteins that contained encapsulation peptides and demonstrated that the shell layer follows discrete rules in its assembly. These results are consistent overall with a model in which biomolecular condensation drives interactions of cargo proteins before envelopment by shell layer proteins.