A near-atomic-resolution structure of J-aggregated helical light-harvesting nanotubes (LHNs) has been determined using cryo-electron microscopy (cryo-EM). The structure of LHNs, derived from an amphiphilic cyanine dye (C8S3-Cl), reveals a brick layer arrangement of chromophores, contradicting the previously hypothesized herringbone arrangement. The structure also identifies a new non-biological supramolecular motif—interlocking sulfonates—that is responsible for the slip-stacked packing and J-aggregate nature of the LHNs. This work demonstrates how independently obtained native-state structures complement photophysical measurements and enable accurate understanding of (excitonic) structure–function properties, informing materials design for light-harvesting chromophore aggregates. The structure shows that the inner wall of the LHNs has a higher resolution due to tighter packing and protection from the solvent environment. The inner wall contains six molecules per asymmetric unit (ASU), with a π-π stacking distance of 3.4 Å and a slip between adjacent molecules of 9.0 Å. The outer wall has eight molecules per ASU, with a helical rise of 9.9 Å and a twist of 33.6°. The structure also reveals a regular crisscrossed pattern of sulfonate groups in the lumen of the inner wall, which stabilize the delocalized positive charge on the chromophores. The structure provides a benchmark for the geometric parameters that excitonic couplings depend on. The results show that the LHNs have a unique photophysical behavior, including a narrow red-shifted spectrum and high superradiance. The structure also reveals that the LHNs have a helical symmetry with a rotational symmetry of C5. The results suggest that the interlocking sulfonates are responsible for the slip-stacked packing geometry that is crucial for the J-aggregate behavior of the LHNs. The study also explores the impact of chemical modifications on the self-assembly of LHNs. The results show that replacing the chlorine atoms with bromine increases the diameter of the double-walled nanotubes while conserving the molecular packing. The results also show that adding an extra carbon in the sulfonate chains can disrupt the sulfonate interlocking, either by inducing more disorder or due to steric effects. The study also shows that the LHNs have a robust optical spectrum despite clear structural differences, suggesting that the optical spectra may not be an ideal basis to test structural models against. Instead, independently obtained high-resolution structures form more reliable benchmarks. The study concludes that the LHNs continue to be a rich model system demonstrating novel excitonic behaviors such as superradiance and light harvesting. The structure also sheds light on new insights into self-assembly itself, that will inform the design of supramolecular materials. The structure providesA near-atomic-resolution structure of J-aggregated helical light-harvesting nanotubes (LHNs) has been determined using cryo-electron microscopy (cryo-EM). The structure of LHNs, derived from an amphiphilic cyanine dye (C8S3-Cl), reveals a brick layer arrangement of chromophores, contradicting the previously hypothesized herringbone arrangement. The structure also identifies a new non-biological supramolecular motif—interlocking sulfonates—that is responsible for the slip-stacked packing and J-aggregate nature of the LHNs. This work demonstrates how independently obtained native-state structures complement photophysical measurements and enable accurate understanding of (excitonic) structure–function properties, informing materials design for light-harvesting chromophore aggregates. The structure shows that the inner wall of the LHNs has a higher resolution due to tighter packing and protection from the solvent environment. The inner wall contains six molecules per asymmetric unit (ASU), with a π-π stacking distance of 3.4 Å and a slip between adjacent molecules of 9.0 Å. The outer wall has eight molecules per ASU, with a helical rise of 9.9 Å and a twist of 33.6°. The structure also reveals a regular crisscrossed pattern of sulfonate groups in the lumen of the inner wall, which stabilize the delocalized positive charge on the chromophores. The structure provides a benchmark for the geometric parameters that excitonic couplings depend on. The results show that the LHNs have a unique photophysical behavior, including a narrow red-shifted spectrum and high superradiance. The structure also reveals that the LHNs have a helical symmetry with a rotational symmetry of C5. The results suggest that the interlocking sulfonates are responsible for the slip-stacked packing geometry that is crucial for the J-aggregate behavior of the LHNs. The study also explores the impact of chemical modifications on the self-assembly of LHNs. The results show that replacing the chlorine atoms with bromine increases the diameter of the double-walled nanotubes while conserving the molecular packing. The results also show that adding an extra carbon in the sulfonate chains can disrupt the sulfonate interlocking, either by inducing more disorder or due to steric effects. The study also shows that the LHNs have a robust optical spectrum despite clear structural differences, suggesting that the optical spectra may not be an ideal basis to test structural models against. Instead, independently obtained high-resolution structures form more reliable benchmarks. The study concludes that the LHNs continue to be a rich model system demonstrating novel excitonic behaviors such as superradiance and light harvesting. The structure also sheds light on new insights into self-assembly itself, that will inform the design of supramolecular materials. The structure provides