Fully automated assignment of methyl resonances of a 36 kDa protein dimer from sparse NOESY data

High-resolution solution-state NMR spectroscopy studies of large proteins typically require uniform deuteration of the system and selective protonation and isotope labelling of methyl groups. Under such circumstances, the assignment of methyl resonances presents a considerable experimental challenge and automation of the process using computational algorithms has been actively sought. Through-space connectivities between the labelled methyl groups can be established through nuclear Overhauser enhancement spectroscopy (NOESY). If a high-resolution structure of the system is available, the sparse connectivity restraints derived from this information enable structure-based methyl resonance assignment. Here, we outline a protocol for full automation of the methyl resonance assignment process using the CYANA software package. We tested the protocol on three-dimensional (3D) ¹³C/¹³C-separated NOESY spectra of a dimer of regulatory chains of aspartate transcarbamoylase (ATCase-r₂). We used CYPICK to detect NOE signals, followed by automatic resonance assignment with FLYA. On this dataset, FLYA generated highly similar results using either automatically or manually generated peak lists, confidently assigning ∼60% of the methyl groups with high accuracy (95 ± 2% correctness). We compared this performance to two alternative automatic methyl assignment protocols, MAP-XSII and FLAMEnGO2.0, both of which, similarly to FLYA, support unassigned NOESY peak lists as input. (paper)