During NMR resonance assignment it is often necessary to relate nuclei to one another indirectly, through their common correlations to other nuclei. Covariance NMR has emerged as a powerful technique to correlate such nuclei without relying on error-prone peak peaking. However, false-positive artifacts in covariance spectra have impeded a general application to proteins. We recently introduced pre- and postprocessing steps to reduce the prevalence of artifacts in covariance spectra, allowing for the calculation of a variety of 4D covariance maps obtained from diverse combinations of pairs of 3D spectra, and we have employed them to assign backbone and sidechain resonances in two large and challenging proteins. In this chapter, we present a detailed protocol describing how to (1) properly prepare existing 3D spectra for covariance, (2) understand and apply our processing script, and (3) navigate and interpret the resulting 4D spectra. We also provide solutions to a number of errors that may occur when using our script, and we offer practical advice when assigning difficult signals. We believe such 4D spectra, and covariance NMR in general, can play an integral role in the assignment of NMR signals.
Tugarinov V, Kay LE (2003) Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc 125:13868–13878. doi:10.1021/ja030345sCrossRefPubMedGoogle Scholar
Blinov KA, Larin NI, Kvasha MP et al (2005) Analysis and elimination of artifacts in indirect covariance NMR spectra via unsymmetrical processing. Magn Reson Chem 43:999–1007. doi:10.1002/mrc.1674CrossRefPubMedGoogle Scholar
Blinov KA, Larin NI, Williams AJ et al (2006) Long-range carbon-carbon connectivity via unsymmetrical indirect covariance processing of HSQC and HMBC NMR data. Magn Reson Chem 44:107–109. doi:10.1002/mrc.1766CrossRefPubMedGoogle Scholar
Snyder DA, Ghosh A, Zhang F et al (2008) Z-matrix formalism for quantitative noise assessment of covariance nuclear magnetic resonance spectra. J Chem Phys 129:1–9. doi:10.1063/1.2975206CrossRefGoogle Scholar
Snyder DA, Xu Y, Yang D, Brüschweiler R (2007) Resolution-enhanced 4D 15N/13C NOESY protein NMR spectroscopy by application of the covariance transform. J Am Chem Soc 129:14126–14127. doi:10.1021/ja075533nCrossRefPubMedGoogle Scholar
Harden B, Nichols S, Frueh D (2014) Facilitated assignment of large protein NMR signals with covariance sequential spectra using spectral derivatives. J Am Chem Soc 136:13106–13109CrossRefPubMedPubMedCentralGoogle Scholar