Treatment of NOE constraints involving equivalent or nonstereoassigned protons in calculations of biomacromolecular structures

Abstract

Two modifications to the commonly used protocols for calculating NMR structures are developed, relating to the treatment of NOE constraints involving groups of equivalent protons or nonstereoassigned diastereotopic protons. Firstly, a modified method is investigated for correcting for multiplicity, which is applicable whenever all NOE intensities are calibrated as a single set and categorised in broad intensity ranges. Secondly, a new set of values for 'pseudoatom corrections' is proposed for use with calculations employing 'centre-averaging'. The effect of these protocols on structure calculations is demonstrated using two proteins, one of which is well defined by the NOE data, the other less so. It is shown that failure to correct for multiplicity when using 'r(-6) averaging' results in overly precise structures, higher NOE energies and deviations from geometric ideality, while failure to correct for multiplicity when using 'r(-6) summation' can cause an avoidable degradation of precision if the NOE data are sparse. Conversely, when multiplicities are treated correctly, r(-6) averaging, r(-6) summation and centre averaging all give closely comparable results when the structure is well defined by the data. When the NOE data contain less information, r(-6) averaging or r(-6) summation offer a significant advantage over centre averaging, both in terms of precision and in terms of the proportion of calculations that converge on a consisten result.