This study presents a 3.3 Å cryo-electron microscopy (cryo-EM) structure of light-harvesting nanotubes (LHNs), which are biomimetic supramolecular aggregates derived from an amphiphilic cyanine dye (C8S3-Cl). The structure reveals a brick layer arrangement of chromophores, revising the previously hypothesized herringbone arrangement. The interlocking sulfonate groups are identified as a new non-biological supramolecular motif responsible for the slip-stacked packing and J-aggregate nature of the LHNs. This work highlights the importance of high-resolution structures in understanding and modulating long-range excitonic coupling, providing insights into the design of materials for light-harvesting applications. The study also explores the impact of chemical modifications on the self-assembly process, demonstrating the sensitivity of LHNs to small changes in molecular structure. The findings contribute to the broader goal of achieving precise control over the structure–function relationships in supramolecular materials.This study presents a 3.3 Å cryo-electron microscopy (cryo-EM) structure of light-harvesting nanotubes (LHNs), which are biomimetic supramolecular aggregates derived from an amphiphilic cyanine dye (C8S3-Cl). The structure reveals a brick layer arrangement of chromophores, revising the previously hypothesized herringbone arrangement. The interlocking sulfonate groups are identified as a new non-biological supramolecular motif responsible for the slip-stacked packing and J-aggregate nature of the LHNs. This work highlights the importance of high-resolution structures in understanding and modulating long-range excitonic coupling, providing insights into the design of materials for light-harvesting applications. The study also explores the impact of chemical modifications on the self-assembly process, demonstrating the sensitivity of LHNs to small changes in molecular structure. The findings contribute to the broader goal of achieving precise control over the structure–function relationships in supramolecular materials.