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

Neutron Macromolecular Crystallography

Locates the positions of critical hydrogen atoms in protein crystals at atomic resolution

Oxidative cleavage of glycosidic bonds. Neutron and X-ray crystallography reveal structural details of the enzymatic action of lytic polysaccharide monooxygenase (green and yellow ribbon diagrams) as it breaks polysaccharide chains (gold). This activity enhances the enzymatic hydrolysis of recalcitrant carbohydrate biomass, such as cellulose or chitin. [From O’Dell, W. B., P. K. Agarwal, and F. Meilleur. 2017. “Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase,” Angew. Chem. Int. Ed. 56, 767–770. DOI: 10.1002/anie.201610502. © 2017 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim.]

Neutron macromolecular crystallography (NMC) provides information about the location of critical hydrogen atoms in protein crystals at atomic resolution. The technique is complementary to X-ray crystallography, which can determine the three-dimensional positioning of other elements in biological macromolecules but not hydrogen. The X-ray photons used in X-ray crystallography interact with the atomic electric field proportional to atomic number, making hydrogen all but invisible to X-rays. In contrast, the neutrons used in NMC interact with atomic nuclei, making it possible to observe hydrogen and deuterium (heavy hydrogen) and distinguish these from heavier elements such as carbon, nitrogen, and oxygen. This feature of NMC enables studies of hydrogen bonding networks and the protonation states of catalytic residues. In addition, neutrons do not cause radiation damage as is often the case with methods that utilize X-rays or electrons. NMC does, however, require significantly larger crystal volumes than X-ray techniques.


Key Features of Neutron Macromolecular Crystallography

  • Nondestructive probe causes no radiation damage to samples.
  • Critical hydrogen atoms can be observed in the active sites of enzymes and hydrogen bonding networks.
  • Ionization states of amino acids can be determined.
  • Redox state of metalloenzymes can be probed.
  • Measurements are typically performed under near-physiological conditions (i.e., room temperature).

BER Researchers Use Neutron Macromolecular Crystallography to Study:

  • The mechanism of biomass-degrading enzymes
  • The redox state of metalloenzymes
  • The binding of ligands to proteins

See more examples in Science Highlights

Sample Considerations

  • Sample proteins must be crystallized.
  • A crystal size of 1 mm3 is needed for hydrogenated proteins.
  • A crystal size of 0.1 mm3 is needed for deuterated proteins.
  • Samples can be measured at room temperature due to the technique’s nondestructive nature.
  • Measurements may also be performed under cryogenic conditions.
  • A complementary X-ray diffraction dataset is needed to refine macromolecular structure.

Neutron Macromolecular Crystallography 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.