Fig. 1: Outline of the BARASA triple resonance assignment algorithm.

a The search engine rests on a Bayesian-based simulated annealing protocol that uses a specific-heat mechanism to guide cooling. Crosspeaks lists drawn from triple resonance spectra are assembled into putative spin systems, which are then randomly assigned to positions within the primary sequence of the protein. Sequential adjacency in the primary sequence is provided by apparent connectivities derived from triple resonance NMR spectra. Predicted chemical shifts, based on a high-resolution structural model or gleaned from empirical amino acid-specific distributions, are incorporated into the system energy using Bayesian statistics. Throughout annealing, crosspeaks may move among spin systems with overlapping resonances, changing the energies of the affected spin systems. Annealing involves Monte Carlo swapping of both crosspeak assignments to spin systems and spin system assignments to locations in the sequence. The concept of dynamic swapping of individual crosspeaks or entire spin systems is outlined in Fig. 2. Annealing continues until energy equilibration is achieved. The temperature is then lowered and the system re-equilibrated. Annealing is stopped when the termination criteria are met and a local minimization routine is performed. b The final resonance assignments are developed from results of multiple independent simulated annealing runs. c Shown is a ribbon representation of maltose binding protein (PDB code: 1DMB [https://doi.org/10.2210/pdb1DMB/pdb]) color-coded according to assignment status following analysis by BARASA: correctly assigned residues (blue); unassigned residues (white), prolines (red). See main text for further details.