Fig. 3. Two major conformations of KcsA selectivity filter from 2D IR simulations for the (top) open-constricted state and (bottom) closed-constricted state.

(A) Calculated 2D IR spectrum of the entire MD trajectory of the open-constricted state reveals a major peak at ω_pump = 1617 cm⁻¹ and a minor peak at ω_pump = 1602 cm⁻¹. Similar to the closed-constricted state analysis (top), two major ion binding configurations are revealed. (B) Spectrum for structural confomation with no K⁺ ion at the S1 binding site, exhibiting a high frequency peak at ω_pump = 1617 cm⁻¹ and constituting 73% of the states observed in the MD trajectory of the open-constricted state. (C) Spectrum for states in which the S1 site is occupied, which accounts for 14% of all the structures in the MD trajectory. The calculated spectrum of this conformation reveals a single low frequency peak at ω_pump = 1602 cm⁻¹. (D) Calculated 2D IR spectrum of the entire MD trajectory of the closed-constricted state reveals a major peak at ω_pump = 1601 cm⁻¹. (E) Simulated spectrum of the structural configuration without a K⁺ ion in S1 shows a high frequency peak at ω_pump = 1617 cm⁻¹. This configuration in 21% of the structural snapshots. (F) The simulated spectrum for the structural sub-ensemble with a K⁺ ion bound at S1, which accounts for 72% of the structural snapshots in the MD trajectory. The major peak has a peak frequency of ω_pump = 1601 cm⁻¹. Dashed lines at ω_pump = 1620 cm⁻¹ (green) and ω_pump = 1600 cm⁻¹ (orange) are shown as guides. All spectra are normalized to the most intense peak.