Flash X-ray diffraction imaging in 3D: a proposed analysis pipeline
This work addresses the time-consuming and complex data processing problem for researchers in structural biology using FXI experiments, but it is incremental as it builds on existing methods with a proposed pipeline.
The authors tackled the challenge of reconstructing 3D electron densities from millions of 2D diffraction patterns in Flash X-ray diffraction Imaging (FXI) by proposing a semi-automatic data analysis pipeline, and they applied it to quantify the 3D electron structure of the PR772 virus, achieving resolution above the detector-edge and clearly showing the pseudo-icosahedral capsid.
Modern Flash X-ray diffraction Imaging (FXI) acquires diffraction signals from single biomolecules at a high repetition rate from X-ray Free Electron Lasers (XFELs), easily obtaining millions of 2D diffraction patterns from a single experiment. Due to the stochastic nature of FXI experiments and the massive volumes of data, retrieving 3D electron densities from raw 2D diffraction patterns is a challenging and time-consuming task. We propose a semi-automatic data analysis pipeline for FXI experiments, which includes four steps: hit finding and preliminary filtering, pattern classification, 3D Fourier reconstruction, and post analysis. We also include a recently developed bootstrap methodology in the post-analysis step for uncertainty analysis and quality control. To achieve the best possible resolution, we further suggest using background subtraction, signal windowing, and convex optimization techniques when retrieving the Fourier phases in the post-analysis step. As an application example, we quantified the 3D electron structure of the PR772 virus using the proposed data-analysis pipeline. The retrieved structure was above the detector-edge resolution and clearly showed the pseudo-icosahedral capsid of the PR772.