BER Structural Biology and Imaging Resources
Synchrotron, Neutron, and Cryo-EM
U.S. Department of Energy | Biological and Environmental Research Program

X-ray Footprinting

Structure determination through probing solvent accessible regions of biomolecules

Left: With the method of X-ray footprinting, an X-ray beam interacts with water in solution to produce hydroxyl radicals, which covalently modify proteins in solvent-accessible regions; these modifications are detected using standard liquid chromatography mass spectrometry (LC-MS). Right: Highlights of the types of structural information that can be obtained using the method, from dynamics of G-protein-coupled receptors (GPCR) activation, to prion surface features, to how proteins fit together in cellular structures.[Image Credits: (1) Lawrence Berkeley National Laboratory; (2) Reprinted from Du, Y., et al. 2019. Assembly of a GPCR-G Protein Complex, Cell 177(5), 1232-42, with permission from Elsevier; (3) Reprinted under a Creative Commons Attribution 4.0 International License (CC BY 4.0) from Sommer, M., et al. 2019. DOI: 10.1104/pp.18.01190; (4) Lawrence Berkeley National Laboratory; (5) Case Western Reserve University.]

 X-ray footprinting is an approach to identifying solvent accessible regions of a macromolecule (protein or nucleic acid). Solvent accessibility can be an indicator of a binding surface within the macromolecule or an area of conformational movement.  When used in combination with other structural biology techniques, footprinting provides complementary or validating information particularly about dynamical motion. In this approach, short pulses of intense synchrotron X-rays are used to produce hydroxyl radicals that can react with the target macromolecule. For proteins, mass spectrometry analysis of the irradiated sample, cleaved by a protease, is the essential follow up step necessary to identify the locations of X-ray–induced irreversible hydroxyl radical formation (which are a proxy for solvent-accessible amino acid residues).  In the case of nucleic acids, hydroxyl radicals will cleave solvent-accessible phosphate backbones, and gels or sequencing-based methods are used to identify segments of the nucleic acid that were not damaged. Identifying solvent-accessible surfaces is a process that has been around for a long time, using chemicals rather than X-rays to generate the hydroxyl radicals within a solution or macromolecule. Another approach is through hydrogen-deuterium exchange experiments. The advantage of using synchrotron X-rays to generate hydroxyl radicals is that the intense brief burst of radiation produces the necessary pulse of radicals for rapid irreversible structural modification, without damaging other structural features. It also has the advantage of requiring a smaller amount of sample than chemical approaches.

Conceptual overview available here:

Key Features of X-ray Footprinting

  • Data obtained is used to construct a “water map,” revealing locations of water molecules at the time of X-ray exposure
  • Typical X-ray exposure time on the order of microseconds or milliseconds
  • Yields protein conformation or protein-protein interaction regions as a function of time
  • Oxidative modifications are permanent and preserved post-exposure so samples can be stored for later analysis by liquid chromatography mass spectrometry (LC-MS) or sequencing, as appropriate to the sample type

BER Researchers Use X-ray Footprinting to Study:

  • Evolution of protein structure and protein-protein interaction in solution under both steady-state or time-resolved conditions, including in response to outside stressors
  • Protein or nucleic acid folding or unfolding mechanisms
  • Protein-nucleic acid interactions
  • Develop structural understanding of intrinsically disordered proteins that are challenging for standard methods
  • Examination of protein or nucleic acids structure in intact viruses or from brain tissue
  • Small molecule interactions with proteins, such as drug binding to targets or binding of proteins to inorganic structures

Sample Considerations

  • 5-10 micromolar concentration
  • 5-200 microliters sample volumes depending on the experiment
  • Sample buffers should be minimally scavenging (e.g., no glycerol)
  • 96-well plate or liquid jet/capillary sample delivery options
  • On-site or remote beamline access; LC-MS on demand

 X-ray Footprinting Beamlines at DOE User Facilities


  • Du, Y., et al. 2019. “Assembly of a GPCR-G Protein Complex,” Cell 177(5), 1232-42. DOI: 10.1016/j.cell.2019.04.022.
  • Sommer, M., et al. 2019. “Heterohexamers Formed by CcmK3 and CcmK4 Increase the Complexity of Beta Carboxysome Shells,” Plant Physiology 179(1), 156-67. DOI: 10.1104/pp.18.01190.
  • Schoof, M., et al. 2020. “An Ultrapotent Synthetic Nanobody Neutralizes SARS-CoV-2 by Stabilizing Inactive Spike,” Science 370(6523), 1473-79. DOI: 10.1126/science.abe3255.
  • Kamali-Jamil, R., et al. 2021. “The Ultrastructure of Infectious L-type Bovine Spongiform Encephalopathy Prions Constrains Molecular Models,” PLoS Pathogens 17(6), e1009628. DOI: 10.1371/journal.ppat.1009628.