Automatic 13C chemical shift reference correction for unassigned protein NMR spectra

Abstract

Poor chemical shift referencing, especially for 13C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct 13C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ± 0.4 ppm of the correct 13C reference value. On a much larger unassigned chemical shift test set, the base method kept 13C chemical shift referencing errors to within ± 0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct 13C chemical shift referencing errors to within ± 0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct 13C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct 13C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.Electronic supplementary materialThe online version of this article (10.1007/s10858-018-0202-5) contains supplementary material, which is available to authorized users.

BaMORC: A Software Package for Accurate and Robust 13C Reference Correction of Protein NMR Spectra

“…Both functions utilize the output from the read-in functions mentioned above and will perform a secondary structure estimation based on the provided protein sequence if secondary structure information is not provided. Through a series of optimization calculations (for details refer to paper 15 ), bamorc() and unassigned_bamorc() will return the estimated referencing correction value in a plain text file and corrected chemical shifts for both C α and C β as a table, as shown in Figure 2. The user can optionally customize the search range.…”

“…As shown in Figure 4, the reference correction component of unassigned BaMORC provides reference correction values within ±0.45 ppm for 90% of the datasets, representing a 90% CI of 0.69 ppm. 15 This suggests that the unassigned BaMORC algorithm can achieve the same level of performance when handling unassigned 13 C protein NMR peak list data. This level of real-world performance is demonstrated with a set of peak lists derived from solution NMR HN(CO)CACB spectra for 10 different proteins.…”

“…With chemical shift assignments, BaMORC provides reference correction values within ±0.50 ppm for all datasets and within ±0.22 ppm for 90% of the datasets, representing a 90% confidence interval (CI) of 0.40 ppm (Figure 3). 15 This level of performance is superior to the prior state-of-the-art linear analysis of chemical shifts (LACS) method. 14 However, in the real-world situation, 13 C reference correction is most valuable before protein resonance assignments are known.…”

“…In this real-world evaluation, unassigned BaMORC provided reference correction values all within ±0.40 ppm. 15…”

“…We therefore developed the Bayesian model optimized reference correction (BaMORC) method 15 that helps non-expert scientists to detect and correct 13 C C α and C β chemical shifts, at the beginning of the protein NMR analysis process, when chemical shifts are unassigned. Here, we describe the BaMORC method implemented in an easy-to-use software package written in the R programming language.…”