Integrating <sup>19</sup>F Distance Restraints for\nAccurate Protein Structure Determination by Magic Angle Spinning NMR\nSpectroscopy

Traditional protein structure determination\nby magic angle spinning\n(MAS) solid-state NMR spectroscopy primarily relies on interatomic\ndistances up to 8 Å, extracted from <sup>13</sup>C-, <sup>15</sup>N-, and <sup>1</sup>H-based dipolar-based correlation experiments.\nHere, we show that <sup>19</sup>F fast (60 kHz) MAS NMR spectroscopy\ncan supply additional, longer distances. Using 4F-Trp,U-<sup>13</sup>C,<sup>15</sup>N crystalline <i>Oscillatoria agardhii</i> agglutinin (OAA), we demonstrate that judiciously designed 2D and\n3D <sup>19</sup>F-based dipolar correlation experiments such as (H)CF,\n(H)CHF, and FF can yield interatomic distances in the 8–16\nÅ range. Incorporation of fluorine-based restraints into structure\ncalculation improved the precision of Trp side chain conformations\nas well as regions in the protein around the fluorine containing residues,\nwith notable improvements observed for residues in proximity to the\nTrp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops,\nwhich lacked sufficient long-range <sup>13</sup>C-<sup>13</sup>C distance\nrestraints. Our work highlights the use of fluorine and <sup>19</sup>F fast MAS NMR spectroscopy as a powerful structural biology tool.