DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β 2 ) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ 3 δδ′ψχ (254kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB 4 ) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ–SSB 4 (108kDa) and the clamp loader–SSB 4 (340kDa) consistent with IM data. A significant increase in measured collision cross-section (~10%) of the clamp loader–SSB 4 complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader–SSB 4 complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.