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.
Highlights
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 startto-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility
We provide an insight into the ionization dynamics and demonstrate how the presence of the water layer influences the resolution-dependent fidelity of reconstructing the protein alone, and discuss its consequences for Single-particle imaging (SPI)
We have explored the effect of water layer thickness on the fidelity of diffraction patterns produced from hydrated proteins in a modeled single-particle imaging experiment
Summary
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 startto-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The orientation of the sample with respect to the beam and the detector is unknown, the individual patterns must be oriented and merged into a three-dimensional diffraction volume (using dedicated algorithms)[7,8] before the three-dimensional electron-density map is reconstructed via phase retrieval[9] These limitations of X-ray FEL imaging to resolving the structure of biologically relevant single macromolecules in a SPI experiment have been discussed in detail by, e.g., Fortmann-Grote et al.[10]. In addition to those challenges, the heterogeneity of the protein sample may limit high resolution single-particle imaging[11,12]. The superconducting accelerator technology enables generating from tens of thousands up to a million light flashes per second, which, in turn, makes it possible to record the required high number of diffraction images within a feasible experiment duration
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