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Time-resolved X-ray solution scattering (TR-XSS) is a synchrotron-based methodology that enables real-time structural characterization under near-native conditions to provide insight into dynamic and transient structural changes inaccessible to static high-resolution methods such as cryo-electron microscopy (cryo-EM) or X-ray crystallography. Here, we present a workflow for light-triggered TR-XSS experiments that spans data collection, data processing, kinetic analysis, and structural refinement, with accompanying Python scripts. A calcium-transporting P-type ATPase membrane protein (LMCA1) is used as an illustrative example, but the protocol is broadly applicable to diverse protein systems. This workflow offers a practical framework for collecting TR-XSS synchrotron data and subsequent data analysis and interpretation.

Time-resolved X-ray solution scattering (TR-XSS) enables tracking of protein structural dynamics but can suffer from radiation damage when performed with continuous X-ray beam exposure at multipurpose small-angle X-ray scattering (SAXS) beamlines. Here, we systematically assess how protein concentration, exposure time, X-ray dose and beam focusing influence the stability of adenylate kinase (AdK) during TR-XSS at CoSAXS at the MAX IV Laboratory. Static SAXS measurements from 2.5 to 21 mg ml−1 showed only minor interparticle effects, establishing suitable concentrations for TR-XSS. Under detector-focused conditions, AdK irradiated at room temperature remained stable up to 3.13 kGy, with consistent absolute and difference scattering profiles over 50 ms. In contrast, focusing the beam directly on the sample increased the absorbed dose to 21.3 kGy and produced clear signatures of radiation damage, which included low-q intensity loss and increasing deviation from the low-dose condition. These results identify practical system-specific dose limits and highlight beam focusing as a key determinant of sample integrity. The workflow presented here provides general guidelines for minimizing radiation artifacts in TR-XSS on multipurpose SAXS beamlines.