The experimental accuracy of the uni-directional exact NOE.

We have established protocols to calculate exact NOEs (eNOE) from NOE data. eNOEs lend unprecedented precision to the calculation of distance restraints used for structure calculation. Moreover, as eNOEs are averaged quantities over all conformations of a molecule, they may contain accessible information of the sampled conformational space. In practice, a prerequisite for an exact interpretation is the evaluation of both NOESY cross-peak buildups. For large molecular sizes, the fraction of NOEs which can only be obtained from one cross peak typically increases. Distance restraints derived from such NOEs must be used with a tolerance for errors associated with the broken symmetry of the individual magnetization transfer pathways. The correct choice of upper and lower limits is particularly important for multiple-state ensemble calculation, where too narrow tolerances may lead to incorrect spatial sampling. In order to dissect these pathways in heavy-atom resolved 3D NOESY experiments, we analyze 2D [(1)H, (1)H]-NOESY experiments, which are the fundamental building blocks of the former. In combination with an analysis of excitation and inversion profiles of pulses on heavy atoms and relaxation effects during HXQC elements, we derive a rule for the correct choice of upper and lower distance limits derived from such uni-directional NOEs. We show that normalization of the cross- to the diagonal-peak intensities of the spins of magnetization destination rather than origin leads to similar errors of the distance restraints. This opens up the prospect of extended collection of unidirectional eNOEs.