Several distinct computational approaches have recently been implemented to represent conformational heterogeneity from time- and space-averaged X-ray crystallography datasets. As these modeling methods mature, newly discovered alternative conformations are being used to derive functional protein mechanisms. Room temperature X-ray data collection is emerging as a key variable for sampling functionally relevant conformations also observed in solution studies. Although concerns about radiation damage are warranted with higher temperature data collection, “diffract and destroy” strategies on X-ray free electron lasers may permit radiation damage-free data collection. X-ray crystallography need not be confined to “static unique snapshots”; these experimental and computational advances are revealing how the many conformations populated within a single crystal are used in biological mechanisms.