Solution X-ray Scattering (SAXS)

Static and dynamic structural information on macromolecules in solution

Left: Solution X-ray scattering (SAXS) enables high-throughput structural characterizations of gene products in solution. The technique can distinguish between aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and structures similar to unknown structures, and determine protein envelopes. Right: SAXS reveals how protein-mediated reorganization of microbial DNA adapts E. coli to a changing environment. Experimental SAXS curves (left) indicate that HU, a histone-like nucleoid-associated protein (blue and cyan structures at right), organizes the structure of the nucleoid (yellow) differently at a high salt concentration (300mM) than at lower concentrations (50-150mM). This, in turn, affects global gene regulation in the cell. [Left: From Hura, G. L., et al. 2009. “Robust, High-Throughput Solution Structural Analyses by Small Angle X-ray Scattering (SAXS),” Nature Methods 6, 606–12. DOI: 10.1038/NMETH.1353.] Right: Reprinted under a Creative Commons Attribution 4.0 International (CC BY 4.0) license from Remesh, S. G., et. al. 2020. “Nucleoid remodeling during environmental adaptation is regulated by HU-dependent DNA bundling,” Nature Communications 11, 2905. DOI: 10.1038/s41467-020-16724-5.]

Solution X-ray scattering provides information about macromolecular structure and dynamics from solutions or partially ordered arrays of biomolecules under physiologically relevant conditions. This type of X-ray scattering is frequently referred to as small angle X-ray scattering (SAXS) because structural information is most often detected at small angles relative to the probing X-ray beam. SAXS captures information at a lower resolution than macromolecular crystallography or cryo-electron microscopy, so most investigators use the technique to characterize macromolecular behavior in solution at the nanometer scale.

SAXS is a powerful tool for time-resolved studies investigating large conformational changes during enzyme reactions, protein-protein interactions, and protein folding intermediates. An advantage to performing SAXS at a synchrotron experimental station is that data can be collected during sample elution from attached purification columns. This enables unique access to transient complexes that rapidly assemble and disassemble and observations of the effects of varied solution conditions on stabilizing complexes or processes.

Preparation of samples in solution for SAXS is relatively simple, enabling high-throughput data collection. In an era where mutations are easily introduced or sequence variants are contrasted, high-throughput screening allows hundreds of constructs to be characterized within hours. SAXS is therefore an ideal assessment tool for use in the iterative design cycle of macromolecular engineering. While structurally homogeneous samples enable the extraction of more detailed structural information than heterogeneous samples, scattering from heterogeneous materials can also be used to characterize changes in the degree of ordering used to monitor, for example, the degradation of cellulose.

Key Features of SAXS

• Rapid processing (ms) of small sample volumes (µL) in solution (almost any condition)
• Data collection during sample purification can increase homogeneity
• Capture of nanometer-scale information in solutions
• High-throughput collection of hundreds of samples in hours
• Data collection at any step of a microfluidic processing scheme

BER Researchers Use SAXS to Study:

• Macromolecular engineering (novel protein design)
• Transient macromolecular complexes (photoprotection in plants)
• Macromolecular structure and dynamics (Rubisco)
• Nanoscale properties of ordered and semi-ordered hierarchical systems (2D protein sheets)
• Time-resolved nanoscale phenomena (conformational changes upon ATP binding)

See more examples in Science Highlights

Sample Considerations

• Microliter volumes of solution at mg/mL concentrations
• More information with homogenous samples
• Greater degree of scattering with electron-rich atoms
• Greater degree of scattering with larger particles
• Requires comparison between solution with and without macromolecule of interest

SAXS Beamlines at DOE User Facilities

Each beamline has unique characteristics. To determine the user facility and beamline best suited to your science questions, see additional information and beamline contacts at the links below.

• Beamline 12.3.1 (SIBYLS) — Advanced Light Source
• Beamlines at Sector 18 (BioCAT) — Advanced Photon Source
• Beamline 16-ID (LiX) — National Synchrotron Light Source II
• Beamline 4-2 (SMB) — Stanford Synchrotron Radiation Lightsource

Research Highlights