Application of a real-space three-dimensional image reconstruction method in the structural analysis of noncrystalline biological macromolecules enveloped by water in coherent x-ray diffraction microscopy

Abstract

Coherent x-ray diffraction microscopy is a novel technique in the structural analyses of particles that are difficult to crystallize, such as the biological particles composing living cells. As water is indispensable for maintaining particles in functional structures, sufficient hydration of targeted particles is required during sample preparation for diffraction microscopy experiments. However, the water enveloping particles also contributes significantly to the diffraction patterns and reduces the electron-density contrast of the sample particles. In this study, we propose a protocol for the structural analyses of particles in water by applying a three-dimensional reconstruction method in real space for the projection images phase-retrieved from diffraction patterns, together with a developed density modification technique. We examined the feasibility of the protocol through three simulations involving a protein molecule in a vacuum, and enveloped in either a droplet or a cube-shaped water. The simulations were carried out for the diffraction patterns in the reciprocal planes normal to the incident x-ray beam. This assumption and the simulation conditions corresponded to experiments using x-ray wavelengths of shorter than 0.03 Å. The analyses demonstrated that our protocol provided an interpretable electron-density map. Based on the results, we discuss the advantages and limitations of the proposed protocol and its practical application for experimental data. In particular, we examined the influence of Poisson noise in diffraction patterns on the reconstructed three-dimensional electron density in the proposed protocol.

XFELs for structure and dynamics in biology

“…(viii) Inner-shell X-ray absorption (Mitzner et al, 2013;Kroll et al, 2016) and emission (Kern et al, 2015) spectra may be collected in synchrony with snapshot X-ray scattering from microcrystals, allowing the chemical and spin states of heavy atoms to be tracked in time through a chemical reaction, in correlation with density maps, using pump-probe or mixing experiments.…”

Data processing software suiteSITENNOfor coherent X-ray diffraction imaging using the X-ray free-electron laser SACLA

“…The ZOCHO subprogram for phase-retrieval calculation has been developed through simulation studies on structure analyses of macromolecules in aqueous environment from single-shot diffraction data (Kodama & Nakasako, 2011;. This subprogram implements the hybrid input-output (HIO) algorithm (Fienup, 1982) in combination with the shrink-wrap (SW) algorithm (Marchesini et al, 2003).…”

KOTOBUKI-1 apparatus for cryogenic coherent X-ray diffraction imaging

“…A subprogram named ZOCHO 48,49 in the SITENNO suite is used for phase retrieval from diffraction amplitude To access the quality of sample, we compare the numbers of diffraction patterns possible to be successfully phase-retrieved (N image ), the number of patterns having ROI intensity larger than a given threshold level (N pattern ), and the number of XFEL pulses (N pulse ). c We define C sym as y) is the intensity in a ROI and I sym (x , y ) is that in the symmetry-related ROI.…”

“…50 The progress of the phase retrieval calculation and the quality of the retrieved projection density are monitored by the γ 51 and R F values. Based on our experiences in CXDI simulation studies, 48,49 we apply parameters controlling the HIO and SW calculations, such as the number of HIO cycles before SWupdate of the support area, the standard deviation of the Gaussian function used in the SW calculations, and the threshold to define support for SW-updated density map used in the next iteration cycle (Table I). Data processing and phase-retrieval calculation are carried out immediately after data collection using the SITENNO suite, which is currently installed in a cluster system composed of 960 cores of Intel(R) Xeon(R) CPU X5690 (3.47 GHz/core) at SACLA during limited beamtime.…”