The amide I doorway modes are visualized on the actin structures in Figure ), where the red to blue colors represent the positive to negative phases of vibrations in each frequency range, and the color depth encodes the vibration amplitude . The structure-frequency correlation in amide I spectrum is resolved, validating our peak assignment in 2D IR spectra. Following this assignment, the discrepancy in the spectra between the filament and the strongly bundled network (Figure ) demonstrates a conformational change upon morphological changes. The growth of the shoulder peak around 1680 cm -1 and the decrease in β -sheet feature in the strongly bundled network indicate a fraction of β -sheets transitioning into loops, causing more disordered actin backbone structures. This finding seems to agree with the reported morphological changes driven by the depletion of PEG, where the bundle structures are found to be more heterogeneous than the pure filaments . The difference in the PSA spectral features provides evidence for conformational changes in the three backbone structures upon bundle formation. The change in the amide I band lineshape is quantified by fitting the PSA spectra to Gaussian profiles with three sub-peaks. The raw spectra, fitted spectra, and three fitted subpeaks are shown in Figure . The fitting parameters are shown in Supplementary Figure . Note that all fittings have the peak center of α-helix fixed, such that the parameters of α-helix are roughly kept the same across all samples. It is worth noting that there are likely to be more than three species of amide I stretch in this spectral region because actin backbone structures can form different numbers of hydrogen bonds with water. Here, we fit the spectra with three Gaussian profiles to avoid overfitting, so that each peak corresponds to one backbone structure, irrespective of the hydrogen-bonding species. The simplified model of the three Gaussian profiles (Figure ) can capture the overall PSA lineshape very well. The PSA interpretation is similar to that of FT IR spectra according to the Beer-Lambert law, but the transition dipole moments of the different amide I modes may be different; thus, it is not feasible to quantify secondary structure fractions based on IR lineshapes alone. Nonetheless, the differences in peak amplitudes and widths among the samples can be used to qualitatively describe the changes in the secondary structure across different morphologies, as well as the heterogeneity in the local environments. The full width at half maximum (FWHM) of the Gaussian peak for α-helix, β -sheet, and loop structures are 30, 18, and 13 cm -1 , respectively. The broader width of α-helix can be assigned to a wider distribution of hydrogen-bond populations, where a larger width indicates multiple populations. The narrower width of β -sheet and loops indicates that they are more inclined to have a single species, for example, the one-hydrogen-bonded configuration, compared to the α-helix. The difference in FWHM between α-helix and β -sheet/loop is approximately 15 cm -1 , which matches well with the observed red shifts of amide I stretch due to hydrogen bonds . Another possible explanation for the broader linewidth is the slowdown of the dynamics in the local environment of α-helix, as discussed below.