Native mass spectrometry meets X-rays for the elucidation of protein structures.

Small-angle X-ray scattering (SAXS) is a powerful and widely used technique for structural biology, providing information about solution structures, i.e., without the need for crystallization or cryogenic conditions. However, its applicability is limited in cases where sample heterogeneity, conformational mixtures, or aggregation are present. While modern BioSAXS routinely employs inline purification (e.g., SEC-SAXS) to address heterogeneity, gas-phase SAXS, enabled by native mass spectrometry as a sample delivery method, offers an even higher degree of population selectivity by providing well-defined and mass-selected ion populations. The principal challenge of this approach is the inherently low particle density in the gas phase. In the present overview, we present simulations of gas-phase SAXS viral nonstructural proteins and capsids and compare them against diffraction patterns from prototypical GroEL. We evaluate the expected scattering signal under experimentally realistic conditions and discuss potential implementations and use cases at both synchrotron radiation sources and X-ray free-electron lasers, thereby outlining regimes in which gas-phase SAXS may become a viable complementary tool for structural studies.