Structural Heterogeneity in Single Particle Imaging Using X-ray Lasers

Abstract

One of the challenges facing single particle imaging with ultrafast X-ray pulses is the structural heterogeneity of the sample to be imaged. For the method to succeed with weakly scattering samples, the diffracted images from a large number of individual proteins need to be averaged. The more the individual proteins differ in structure, the lower the achievable resolution in the final reconstructed image. We use molecular dynamics to simulate two globular proteins in vacuum, fully desolvated as well as with two different solvation layers, at various temperatures. We calculate the diffraction patterns based on the simulations and evaluate the noise in the averaged patterns arising from the structural differences and the surrounding water. Our simulations show that the presence of a minimal water coverage with an average 3 Å thickness will stabilize the protein, reducing the noise associated with structural heterogeneity, whereas additional water will generate more background noise.

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

“…This work extends the focus of previous works which investigated specific aspects of imaging (e.g. X-ray triggered sample dynamics 12 , 20 , 21 , 23 , effect of water layer on diffraction patters of undamaged proteins 22 , effect of structural heterogeneity on diffraction patterns 12 ). In this paper, we study single-particle imaging of a hydrated protein for the parameters of the European XFEL, performing a detailed analysis with the start-to-end simulation platform.…”

“…That is, we consider a sample surrounded by a layer of water at the time of exposure to an ultrabright X-ray beam. This idea has been proposed by Hau-Riege et al 20 for single-particle diffraction imaging and further explored in earlier works 12 , 21 – 23 . Ideally , if an object of interest is covered by a (sufficiently thick) tamper layer of another material, this may slow the expansion of the object during its imaging.…”

“…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 also limit high resolution single-particle imaging 11 , 12 . Previous studies 13 have shown that a water tamper layer inhibits protein unfolding, which may occur in vacuum environments common in X-ray instruments.…”

“…Previous studies 13 have shown that a water tamper layer inhibits protein unfolding, which may occur in vacuum environments common in X-ray instruments. Further more, such a layer may also serve to keep the protein sample in a more restricted range of conformations 12 , which in turn could be expected to ease analysis.…”

Reproducibility in the unfolding process of protein induced by an external electric field

“…Diffraction patterns from a single frame at time t from any given simulation trajectory were calculated using an in-house code developed by Martin 41 and used in our previous work. 42,43 It evaluates the pixel intensities I(q,t) forming the pattern as…”