Abstract <p>Rhodopsins are photosensitive transmembrane proteins found in bacteria, archaea, and eukaryotes. Upon photon absorption, they undergo conformational changes critical for their physiological functions, such as ion transport and signal transduction. Microbial rhodopsins are key tools in optogenetics, with applications in biomedical research and clinical treatments. To advance optogenetic techniques, it is crucial to understand the molecular mechanisms of the rhodopsin photocycle. The key approach for studying these mechanisms, cryotrapping, has a setback associated with possible deterioration in resolution due to the unexpectedly large light-induced structural changes. To estimate the scale of these changes, small-angle X-ray scattering (SAXS) can be used. In this study, we applied SAXS to the investigation of light-induced structural changes in purple membranes (PMs) as their two-dimensional crystal organization makes them a suitable model object for studying light-induced changes in 3D rhodopsin crystals. SAXS data from illuminated and non-illuminated PM samples revealed detectable changes, including variations in membrane thickness and planar unit cell dimensions. Particularly, we observed a statistically significant increase in the radius of gyration for flat particles (<i>R</i>ₜ) of ~0.2 Å for the PMs with mutant <i>Hs</i>BR_E204Q, while no significant change was detected for wild-type <i>Hs</i>BR. However, wild-type <i>Hs</i>BR data showed a statistically significant shift in the (1, 1) Bragg peak position (Δ <i>q</i> ~ –10^–4 Å^–1; ~3% of the peak width) upon illumination. For <i>Hs</i>BR_E204Q data, the shift was two times greater; however, in this case due to the lower sample concentration ε_Δq was ~50%, making it difficult to compare the wild-type and mutant cases. Future experiments should aim for better signal-to-noise ratios using synchrotron radiation, higher sample concentrations, and longer exposure times.</p>