Large-scale interdomain rearrangements are essential to protein function, governing the activity of large enzymes and molecular machineries. Yet, obtaining an atomic-resolution understanding of how the relative domain positioning is affected by external stimuli is a hard task in modern structural biology. Here, we show that combining structural modeling by AlphaFold2 with coarse-grained molecular dynamics simulations and NMR residual dipolar coupling data is sufficient to characterize the spatial domain organization of bacterial enzyme I (EI), a ∼130 kDa multidomain oligomeric protein that undergoes large-scale conformational changes during its catalytic cycle. In particular, we solve conformational ensembles for EI at two different experimental temperatures and demonstrate that a lower temperature favors sampling of the catalytically competent closed state of the enzyme. These results suggest a role for conformational entropy in the activation of EI and demonstrate the ability of our protocol to detect and characterize the effect of external stimuli (such as mutations, ligand binding, and post-translational modifications) on the interdomain organization of multidomain proteins. We expect the ensemble refinement protocol described here to be easily transferrable to the investigation of the structure and dynamics of other uncharted multidomain systems and have assembled a Google Colab page (https://potoyangroup.github.io/Seq2Ensemble/) to facilitate implementation of the presented methodology elsewhere.