Protein aggregation represents a bottleneck in the generation and development of antibody-based therapeutics, with a large proportion of candidates failing in formulation (1). It is generally believed that the aggregation propensity of larger antibody reagents, such as immunoglobulin G and Fab, are mostly determined by their variable domain components, although there is currently little understanding of the mechanisms involved (1, 2). Indeed, significant differences of aggregation rates have been reported for antibodies that differ exclusively in their variable domains (1, 2). Aggregation propensity is even more pronounced for smaller antibody reagents, which lack the inter-domain stabilization of their larger counterparts (3, 4). This is a major problem in biotechnology due to an increasing trend towards smaller antibody formats for imaging and tumor targeting applications (4). Common formats include human single chain fragments (scFv) and human single domain antibodies, both of which frequently display poor biophysical properties (1, 3). Here, we present a structural and dynamical insight into the basis of recently identified mutations that prevent the aggregation of human variable light domain.