Validation of macromolecular flexibility in solution by small-angle X-ray scattering (SAXS)

Abstract

The dynamics of macromolecular conformations are critical to the action of cellular networks. Solution X-ray scattering studies, in combination with macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR), strive to determine complete and accurate states of macromolecules, providing novel insights describing allosteric mechanisms, supramolecular complexes, and dynamic molecular machines. This review addresses theoretical and practical concepts, concerns, and considerations for using these techniques in conjunction with computational methods to productively combine solution-scattering data with high-resolution structures. I discuss the principal means of direct identification of macromolecular flexibility from SAXS data followed by critical concerns about the methods used to calculate theoretical SAXS profiles from high-resolution structures. The SAXS profile is a direct interrogation of the thermodynamic ensemble and techniques such as, for example, minimal ensemble search (MES), enhance interpretation of SAXS experiments by describing the SAXS profiles as population-weighted thermodynamic ensembles. I discuss recent developments in computational techniques used for conformational sampling, and how these techniques provide a basis for assessing the level of the flexibility within a sample. Although these approaches sacrifice atomic detail, the knowledge gained from ensemble analysis is often appropriate for developing hypotheses and guiding biochemical experiments. Examples of the use of SAXS and combined approaches with X-ray crystallography, NMR, and computational methods to characterize dynamic assemblies are presented.

Structural insights into the interaction of IL-33 with its receptors

“…In BIL-BOMD, 10,000 distinct conformers were generated by constrained molecular dynamics (MD) simulation, theoretical scattering profiles were calculated by FoXS (37), and then Minimal Ensemble Search (MES) were applied to select a subset containing one to four conformers that best fit the experimental data (36). The full-atomic model is crucial to match the experimental data (38); therefore, missing loops and residues in ST2 and IL-1RAcP ligand-bound crystal structures were constructed with MODELLER (39). For ST2, the theoretically calculated SAXS profile from initial model does not agree well with experimental data (χ = 5.0) (Fig.…”

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding

“…Transformation of the SAXS data exhibited a plateau in the q 3 ⅐I(q) plot and lack of plateau in the q 4 ⅐I(q) plot (Fig. 5, c and d, respectively), indicating that structures were folded yet contained a flexible domain (31,32). Indeed, the low resolution envelope generated via ab initio reconstructions (Fig.…”

The Structure of Human Apolipoprotein A-IV as Revealed by Stable Isotope-assisted Cross-linking, Molecular Dynamics, and Small Angle X-ray Scattering

“…8e). A plateau in this plot is suggestive of intrinsic flexibility in the protein (49,50). This should not be mistaken for an unstructured protein because the q 2 versus q 2 ⅐I(q) plot displays a distinct decrease and not a plateau (Fig.…”