Effects of radiation damage and inelastic scattering on single-particle imaging of hydrated proteins with an X-ray Free-Electron Laser

We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase–Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins.