Reproducibility of protein x-ray diffuse scattering and potential utility for modeling atomic displacement parameters

Abstract

Protein structure and dynamics can be probed using x-ray crystallography. Whereas the Bragg peaks are only sensitive to the average unit-cell electron density, the signal between the Bragg peaks—diffuse scattering—is sensitive to spatial correlations in electron-density variations. Although diffuse scattering contains valuable information about protein dynamics, the diffuse signal is more difficult to isolate from the background compared to the Bragg signal, and the reproducibility of diffuse signal is not yet well understood. We present a systematic study of the reproducibility of diffuse scattering from isocyanide hydratase in three different protein forms. Both replicate diffuse datasets and datasets obtained from different mutants were similar in pairwise comparisons (Pearson correlation coefficient ≥0.8). The data were processed in a manner inspired by previously published methods using custom software with modular design, enabling us to perform an analysis of various data processing choices to determine how to obtain the highest quality data as assessed using unbiased measures of symmetry and reproducibility. The diffuse data were then used to characterize atomic mobility using a liquid-like motions (LLM) model. This characterization was able to discriminate between distinct anisotropic atomic displacement parameter (ADP) models arising from different anisotropic scaling choices that agreed comparably with the Bragg data. Our results emphasize the importance of data reproducibility as a model-free measure of diffuse data quality, illustrate the ability of LLM analysis of diffuse scattering to select among alternative ADP models, and offer insights into the design of successful diffuse scattering experiments.

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

“…We obtained a 1.3-Å resolution XFEL structure of resting G150T ICH at 298 K using 20-to 30-μm microcrystals delivered via a microfluidic electrokinetic sample holder (MESH) injector at the Macromolecular Femtosecond Crystallography (MFX) beamline at LCLS (see Methods). The refined XFEL structure of G150T ICH superimposes nearly exactly [0.14-Å all-atom root mean square deviation (RMSD)] with previous 1.1-to 1.2-Å resolution 274 K synchrotron structures of this mutant protein (48), as expected. However, there are surprising differences between the XFEL and synchrotron structures evident in F o (synchrotron)-F o (XFEL) isomorphous difference electron density maps, which contain over 300 peaks at 3.2σ (0.2 e − /Å 3 ) or higher within 5 Å of the protein and ordered solvent model (Fig.…”

Scaling and merging macromolecular diffuse scattering with mdx2

“…Moreover, in reciprocal space the signals from the short-range and long-range correlations are superimposed, and thus precise measurements are required in order to disentangle the signals from protein motions of interest . Techniques to improve accuracy and precision have been developed, including experimental background measurement and subtraction (Pei et al, 2023), procedures to correct for systematic errors during scaling (Peck et al, 2018;Meisburger et al, 2020) and the development of new statistical indicators of data quality (Su et al, 2021) and model-data agreement .…”

Linking B-factor and temperature-induced conformational transition

“…The B-factor – often called temperature factor [17–20] - relates to the mean-square atomic displacement ( X 2 ) by the Debye-Waller function where X 2 embodies additive crystal contributions from atomic fluctuation ( X 2 A ), conformational motions ( X 2 C ), rigid body vibration ( X 2 RB ) and crystal lattice disorders ( X 2 LT ) [21]. The crystallographic B-factor is also susceptible to variables from crystal (such as hydration, cosolutes, cryoprotection), XRD setup (such as beam, detector), gas stream for temperature control and diffuse scattering [14]. Diffraction shows contributions from both ordered lattice and diffuse scattering [22–25], while diffuse scattering can show contributions from varying sources including rigid-body motion [26] and background scattering [14].…”

“…The crystallographic B-factor is also susceptible to variables from crystal (such as hydration, cosolutes, cryoprotection), XRD setup (such as beam, detector), gas stream for temperature control and diffuse scattering [14]. Diffraction shows contributions from both ordered lattice and diffuse scattering [22–25], while diffuse scattering can show contributions from varying sources including rigid-body motion [26] and background scattering [14]. For these reasons, direct correlations between B-factor with protein dynamics and consequent propensity for conformational changes are still controverse [27].…”

“…, and holds an outstanding position in structural biology for being a highly reproducible technique [13][14][15] .…”