How motions in enzymes might be linked to catalytic function is of considerable general interest. Recent advances in X-ray crystallography and cryogenic electron microscopy offer the promise of elucidating functionally relevant motions in proteins that are not easily amenable to study by other biophysical methods. Here we use 3D variability analysis (3DVA) on cryo-EM maps for wild type (WT) human asparagine synthetase (ASNS) and the R142I ASNS variant to identify conformational changes in the Arg-142 side chain, which mediates the formation of a catalytically relevant intramolecular tunnel. Our 3DVA results for WT ASNS are consistent with independent molecular dynamics (MD) simulations on a model generated from the X-ray structure of human ASNS. Moreover, MD simulations of computational models for the ASNS/β-aspartyl-AMP/MgPPi and R142I/β-aspartyl-AMP/MgPPi ternary complexes, suggest that the structural integrity of the tunnel is impaired in the R142I variant when β-aspartyl-AMP is present in the synthetase active site. The kinetic properties of the R142I ASNS variant support the proposed function of Arg-142. These studies illustrate the power of cryo-EM to identify localized motions and dissect the conformational landscape of large proteins. When combined with MD simulations, 3DVA is a powerful approach to understanding how conformational dynamics might regulate function in multi-domain enzymes possessing multiple active sites.