Fig. 3. Formation of thioimidate intermediate selects catalytically competent active site conformations and redistributes the G150T ICH ensemble.

(A) The 1.3-Å resolution 2mF_o-DF_c XFEL electron density contoured at 0.9σ (blue) for the free G150T ICH at 298 K. Alternate conformations for active site residues are blue (conformer A) and gold (conformer B). (B) The 1.3-Å resolution 2mF_o-DF_c XFEL electron density contoured at 0.9σ (blue) for the thioimidate intermediate observed 30 s after mixing microcrystals with p-NPIC substrate in a mix-and-inject serial crystallography (MISC) experiment. Only conformer A of Cys¹⁰¹ reacts with the intermediate, which redistributes the conformational disorder in the I152 loop. (C) Redistribution of the populations of the active site ensemble upon formation of the thioimidate intermediate. The F_o(30 s)-F_o(0 s) isomorphous difference electron density map is contoured at 3σ (green, positive/red, negative). Arrows show the direction of population shift, from red (loss of population) to green (gain of population). A partially occupied water (red sphere) enters the active site upon formation of the intermediate.