Protein NMR pp 353-373 | Cite as

Covariance NMR Processing and Analysis for Protein Assignment

Part of the Methods in Molecular Biology book series (MIMB, volume 1688)


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.

Key words

NMR Covariance Resonance assignment Peak lists Spectral derivative 4D spectra 



The Frueh lab is supported by the National Institute of Health, grant R01GM104257.


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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Biophysics and Biophysical ChemistryJohns Hopkins University School of MedicineBaltimoreUSA

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