Molecular-level observation of the self-assembly of a virus-like particle.

Biomolecular assembly is a cornerstone of cellular organization. Revealing its underlying principles is essential for understanding biological function1,2 and malfunction in disease3,4. Viral capsid assembly is the archetypal self-assembly system5-7, which has been central in establishing the fundamental principles underpinning biomolecular assembly and the development of new biomaterials8-10 and therapeutics11,12. Yet, despite decades of experimental efforts, observation and quantification of virus self-assembly pathways and dynamics have remained elusive13. Here we combine mass photometry (MP)14 with a single-molecule trapping method to monitor the real-time assembly of individual virus-like particles (VLPs) with molecular resolution. We show that weak and reversible multivalent interactions control the assembly process by facilitating stochastic selection of a limited set of on-path, topologically closed intermediate structures. Assembly is finely tuned by the transition rates between these intermediates, proceeding through a sequence of effectively irreversible first-passage events. The corresponding first-passage times arise from the VLP symmetry, creating temporal separation between the formation of the first topologically closed intermediate and subsequent elongation. This results in a nucleation-and-growth mechanism that yields an equilibrium distribution consistent with the law of mass action, despite the overall irreversibility of assembly. Characterization of the thermodynamics and kinetics of the process reveals how the system specifically assembles into one final structure with high fidelity despite thousands of available assembly intermediates. More broadly, our approach provides a general framework for visualizing and quantifying the dynamics of multimeric biological machines at the molecular level.