Spectral calculations are based on filament structures determined by cryo-electron microscopy (PDB file: 6BNO) to further interpret the IR features of actin and validate the spectral assignment. The excitonic model reproduces the three main experimentally observed modes very well, allowing us to perform further analysis to explain the origin of the modes. However, it does not capture the elongation along the diagonal (Figure ), which results from the static inhomogeneity of the ensemble. To correct for this, we average 10 iterations with random local-mode frequencies drawn from a normal distribution, which results in a close match to the experimental spectra (Figure ). The diagonal traces of the computed 2D IR spectra are extracted to capture the main features of the lineshape (shaded areas in Supplementary Figure ), resulting in three similar main modes that are observed in the experimental spectra as shaded in Figure . Note that PSA analysis is also performed for the calculated Table . The secondary structure contributions to the amide I normal modes are resolved in the six frequency ranges derived from the doorway state analysis. Secondary structure assignments are obtained from the PDB file 6BNO. For simplicity, all non-helical and non-sheet residues are defined as loop structures. 2D IR, which does not resolve the three main modes clearly, likely because the anharmonicity between the two transitions and the computed inhomogeneous broadening in 2D IR are not strictly comparable to the experiments. In Figure , the stick spectrum that indicates the normal-mode intensities is overlaid with the experimental filament PSA lineshape, showing a qualitative match between the experiment and the computation. We apply a doorway state analysis to the calculated eigenmodes to visualize the backbone structures that contribute to different frequency ranges in the amide I region. As described in the Methods section, we divide the region into six frequency ranges, following the work of Chung & Tokmakoff . The ranges R1 to R6 are shaded in gradient colors (light to dark gray) in Figure ). The contributions from three types of secondary structures, as defined in the 6BNO PDB, are calculated for each frequency region and listed in Table . It is observed that R1 contains contributions almost exclusively from β -sheet, when the R5 to R6 range has the most features of loop structures. The contribution from α-helix is spread out over the entire range but is the most dominant in R3.