The conditions for accurately determining distance constraints from TrNOESY data on a small ligand (3′CMP) bound to a small protein (RNase A, <14 kDa) are described. For small proteins, normal TrNOESY conditions of 10:1 ligand:protein or greater can lead to inaccurate structures for the ligand-bound conformation due to the contribution of the free ligand to the TrNOESY signals. By using two ligand:protein ratios (2:1 and 5:1), which give the same distance constraints, a conformation of 3′CMP bound to RNase A was determined (glycosidic torsion angle, χ = -166°; pseudorotational phase angle, 0° ≤ P ≤ 36°). Ligand-protein NOESY cross peaks were also observed and used to dock 3′CMP into the binding pocket of the apo-protein (7rsa). After energy minimization, the conformation of the 3′CMP:RNase A complex was similar to the X-ray structure (1rpf) except that a C3′-endo conformation for the ribose ring (rather than C2′-exo conformation) was found in the TrNOESY structure. © 2007 Elsevier Inc. All rights reserved.