title: On the problem of resonance assignments in solid state NMR of uniformly ¹⁵N,¹³C-labeled proteins.
abstract: Determination of accurate resonance assignments from multidimensional chemical shift correlation spectra is one of the major problems in biomolecular solid state NMR, particularly for relative large proteins with less-than-ideal NMR linewidths. This article investigates the difficulty of resonance assignment, using a computational Monte Carlo/simulated annealing (MCSA) algorithm to search for assignments from artificial three-dimensional spectra that are constructed from the reported isotropic (15)N and (13)C chemical shifts of two proteins whose structures have been determined by solution NMR methods. The results demonstrate how assignment simulations can provide new insights into factors that affect the assignment process, which can then help guide the design of experimental strategies. Specifically, simulations are performed for the catalytic domain of SrtC (147 residues, primarily β-sheet secondary structure) and the N-terminal domain of MLKL (166 residues, primarily α-helical secondary structure). Assuming unambiguous residue-type assignments and four ideal three-dimensional data sets (NCACX, NCOCX, CONCA, and CANCA), uncertainties in chemical shifts must be less than 0.4 ppm for assignments for SrtC to be unique, and less than 0.2 ppm for MLKL. Eliminating CANCA data has no significant effect, but additionally eliminating CONCA data leads to more stringent requirements for chemical shift precision. Introducing moderate ambiguities in residue-type assignments does not have a significant effect.
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA. Electronic address: robertty@mail.nih.gov.
authors: Robert
authors: R
authors: Tycko
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002247:Carbon Isotopes / Q000032:analysis / Q000737:chemistry; D003198:Computer Simulation; D007553:Isotope Labeling; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins
keywords: Automated resonance assignments; Monte Carlo algorithm; Solid state NMR