The protein barnase folds from the denatured state into its native conformation via a high-energy intermediate. Using PhiI-values determined experimentally from single-point mutations as restraints in all-atom molecular dynamics simulations, we have determined ensembles of structures corresponding to the transition states for the formation of the folding intermediate and its conversion into the native state. We have also introduced a stringent validation of the approach used to calculate such structures by predicting interaction PhiIJ-values determined experimentally from a series of double-mutant cycles. The ensembles that we have obtained illustrate the heterogeneity in the nucleation-condensation process by which barnase folds. Obligatory steps of this process include the sequential formation of two folding nuclei, which correspond to the two main hydrophobic cores of the protein. Nonobligatory steps include the elongation of the strand beta1 and the formation of the helix alpha2. The results confirm that the use of experimental observables such as PhiI-values as restraints in molecular dynamics simulations is a powerful general strategy to characterize the relatively heterogeneous structural ensembles that populate nonnative regions of the energy landscapes of proteins.