NMR data are collected as time- and ensemble-averaged quantities. Yet,
in commonly used methods for structure determination of biomolecules,
structures are required to satisfy simultaneously a large number of c
onstraints. Recently, however, methods have been developed that allow
a better fit of the experimental data by the use of time- or ensemble-
averaged restraints. Thus far, these methods have been applied to stru
cture refinement using distance and J-coupling restraints. In this pap
er, time and ensemble averaging is extended to the direct refinement w
ith experimental NOE data. The implementation of time- and ensemble-av
eraged NOE restraints in DINOSAUR is described and illustrated with ex
perimental NMR data for crambin, a 46-residue protein. Structure refin
ement with both time- and ensemble-averaged NOE restraints results in
lower R-factors, indicating a better fit of the experimental NOE data.