build-up rates of the NOE. Inter-proton distances can be determined from unambiguously assigned, well-resolved, high signal-to-noise NOESY spectra by analysis of cross peak intensities. These may be obtained by volume integration and can be converted into estimates of interproton distances. The distance between two atoms and can be calculated from the cross-peak volumes and a scaling constant where can be determined based on measurements of known fixed distances. The range of distances can be reported based on known distances and volumes in the spectrum, which gives a mean and a standard deviation , a measurement of multiple regions in the NOESY spectrum showing no peaks, i.e. noise , and a measurement error . The parameter is set so that all known distances are within the error bounds. This shows that the lower range of the NOESY volume can be shown and that the upper bound is Such fixed distances depend on the system studied. For example, locked nucleic acids have many atoms whose distance varies very little in the sugar, which allows estimation of the glycosidic torsion angles, which allowed NMR to benchmark LNA molecular dynamics predictions. RNAs, however, have sugars that are much more conformationally flexible, and require wider estimations of low and high bounds. In protein structural characterization, NOEs are used to create constraints on intramolecular distances. In this method, each proton pair is considered in isolation and NOESY cross peak intensities are compared with a reference cross peak from a proton pair of fixed distance, such as a geminal methylene proton pair or aromatic ring protons. This simple approach is reasonably insensitive to the effects of spin diffusion or non-uniform correlation times and can usually lead to definition of the global fold of the protein, provided a sufficiently large number of NOEs have been identified. NOESY cross peaks can be classified as strong, medium or weak and can be translated into upper distance restraints of around 2.5, 3.5 and 5.0 Å, respectively. Such constraints can then be used in molecular mechanics optimizations to provide a picture of the solution state conformation of the protein. Full structure determination relies on a variety of NMR experiments and optimization methods utilizing both chemical shift and NOESY constraints. Heteronuclear NOE Some Experimental Methods Some examples of one and two-dimensional NMR experimental techniques exploiting the NOE include: NOESY, Nuclear Overhauser Effect Spectroscopy HOESY, Heteronuclear Overhauser Effect Spectroscopy ROESY, Rotational Frame Nuclear Overhauser