Structure determination relaxation matrix derived distance

To date, only two applications of complete relaxation matrix derived distance restraints have been reported for RNA. MARDIGRAS provided "precise" distance restraints for the structure determination of a 17mer RNA with a flexible loop (26) and a 31mer hairpin with a large internal loop (18). For the latter, assignments and NOE volume extraction was made feasible through deuteration of all cytosines. 

Another principal difficulty is that the precise effect of local dynamics on the NOE intensity cannot be determined from the data. The dynamic correction factor [85] describes the ratio of the effects of distance and angular fluctuations. Theoretical studies based on NOE intensities extracted from molecular dynamics trajectories [86,87] are helpful to understand the detailed relationship between NMR parameters and local dynamics and may lead to structure-dependent corrections. In an implicit way, an estimate of the dynamic correction factor has been used in an ensemble relaxation matrix refinement by including order parameters for proton-proton vectors derived from molecular dynamics calculations [72]. One remaining challenge is to incorporate data describing the local dynamics of the molecule directly into the refinement, in such a way that an order parameter calculated from the calculated ensemble is similar to the measured order parameter. 

The tubulin-bound conformation of DDM in solution has been determined from tr-NOE data [112], Sample conditions were similar to those used previously to determine the bioactive conformation of EpoA. Distance restraints were obtained from a series of tr-NOE spectra recorded at several mixing times and were used in the structure calculation based on the complete relaxation matrix methodology [37], The NMR-derived bioactive conformation is quite similar to the crystal structure except for the conformation of the 8 lactone ring, that is close to a flattened chair in solution but a twisted boat in the crystal (Fig. 18). 

In this context, it is also worth noting that alternative methods exist for quantitative comparisons to NOESY intensities, based mainly on iterative refinements of the rate matrix itself. These models provide a translation from observed intensities to cross-relaxation rates, providing a useful intermediate step in the generation of structural and dynamic models that fit observed data. Much work remains to be done to determine the best approach to refinement and the benefits of going beyond the more common conventional level of structure determinations that uses distance bounds derived by empirical calibration from short-mixing time experiments. 

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