Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that ¹³C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The ¹³C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3′-endo (N) conformation when bound with high affinity to NKA in the E₁Na state, in contrast to the C2′-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H–C–C–H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand–protein interactions.