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

Abstract

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.

Expected resolution limits of x-ray free-electron laser single-particle imaging for realistic source and detector properties

“…S1). We generate each diffraction pattern with a random orientation following a uniform distribution over the SO(3) rotation group 25,43,47 and convert the number of photons to keV units. Then, the Poisson noise is added to diffraction patterns first, and the two AGIPD detector noises are independently added by recalculating the value of each pixel, assuming that each pixel follows a Gaussian distribution scaled to 6 keV, similar to that in the histograms ( Fig.…”

“… 17,20–23 Other obstacles to reach higher resolution are the heterogeneity of samples, 24 weak scattering from biomolecules, and the effects of surrounding sample solvent. 25 Enhancing the spatial resolution in bio-imaging by employing strongly scattering reference objects 26–33 has been discussed and addressed as an alternative option; however, it is non-trivial to apply to 3D SPI experiments. 26 To improve the spatial resolution of SPI at XFELs, single frame signal to background must be improved.…”

Water layer and radiation damage effects on the orientation recovery of proteins in single-particle imaging at an X-ray free-electron laser

“…Several issues posing significant challenges to achieve higher spatial resolution in SPI experiments, such as sample heterogeneity (including sample solvent) 10 , 11 , sample delivery 12 , detector noise 13 and radiation damage 14 , 15 , have been identified 16 . The issues of the sample solvent and radiation damage are interdependent, as the sample solvent can influence the radiation damage of a protein sample and, thus, its effect on the resulting diffraction pattern.…”

“…Water is a common solvent for bio-molecules and is expected to remain attached as a thin layer around the sample even after most of it has evaporated when using the electrospray technique to deliver the sample to an X-ray beam 11 , 18 . The impact of water layer thickness on SPI experiments has been previously investigated by evaluating the features of diffraction patterns from hydrated single particles 11 , 15 , 19 , 20 . However, the combined effect of the radiation damage and water layer thickness on the complete orientation recovery process 21 was not fully addressed in the previous studies.…”

Tree-Code Based Improvement of Computational Performance of the X-ray-Matter-Interaction Simulation Tool XMDYN

“…The SIMEX platform has been used in [8] to estimate the optimal pulse duration for an X-ray pulse of 5 keV photon energy (feasible at the SPB/SFX instrument of the European XFEL) to image reproducible, biological molecules. The platform has also been recently used in [17] to study imaging of hydrated proteins.…”