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.
Highlights
Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins (Saitô 1986; Spera and Bax 1991; Wishart et al 1991; Iwadate et al 1999; Wishart and Case 2001; Neal et al 2003; Mao et al 2011; Serrano et al 2012; Rosato et al 2012)
Since the structural and dynamic information contained in the chemical shift is subtle, even small chemical shifts errors due to inaccurate referencing may provide a distorted representation of the protein, especially if the chemical shifts are directly used in structure determination (Wu et al 2010; Yang and; Bax 2010; Barette et al 2011; Lange et al 2012)
Bayesian Model Optimized Reference Correction (BaMORC) demonstrates robust performance, keeping the 13C reference correction within ± 0.6 ppm at the 90% confidence level even with up to 50% of the 13C chemical shift data missing
Summary
Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins (Saitô 1986; Spera and Bax 1991; Wishart et al 1991; Iwadate et al 1999; Wishart and Case 2001; Neal et al 2003; Mao et al 2011; Serrano et al 2012; Rosato et al 2012). Variance in chemical shifts can be caused by a variety of experimental factors, including pH, temperature, salts, organic solvent mixtures, and inaccurate referencing due to human error (Nowick et al 2003; Ulrich et al 2008). All downstream analyses and interpretations are affected by these inaccuracies in chemical shifts, including the assignment of resonances in biomacromolecules such as proteins. These inaccuracies can outright prevent data analysis, especially with semiautomated data analysis tools, or propagate through data analysis, snowballing into interpretive errors about structure and dynamics. Since the structural and dynamic information contained in the chemical shift is subtle, even small chemical shifts errors due to inaccurate referencing may provide a distorted representation of the protein, especially if the chemical shifts are directly used in structure determination (Wu et al 2010; Yang and; Bax 2010; Barette et al 2011; Lange et al 2012)
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