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

Molecular Structure

The following are imaging and characterization techniques that can be used to study molecular structure.

Cryo-Electron Microscopy and Tomography

Using electrons, cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) techniques provide images of biological materials that are frozen in their native state, ranging from proteins to very large biological assemblies and complexes, at variable resolutions that stretch to the atomic level. Studies are done in two- and three dimensions and do not require crystalline materials.

Neutron Macromolecular Crystallography

Neutron MC provides information about critical hydrogen (H) locations in protein crystals at atomic resolution, complementary to X-ray diffraction, described above. Because X-ray photons interact with the atomic electric field proportional to the atomic number, H is all but invisible to X-rays. In contrast, neutrons interact with nuclei, making it possible to observe H and deuterium (D) and distinguish these light elements next to heavy ones. Hence, studies of H bonding networks and protonation states of catalytic residues are feasible. In addition, neutrons do not cause radiation damage as is often the case with X-rays and electrons. This technique requires significantly larger crystal volumes compared to X-rays.

Small-Angle Neutron Scattering

Like SAXS, SANS is used to study ensemble structures of biological materials with a wide range of length scales in any morphology. SANS, however, can take advantage of the very different neutron scattering cross-sections of H and D, enabling users to selectively highlight different components within a complex system. In combination with H2O/D2O contrast variation and D-labeling, SANS provides unique information about complexes of biomolecules and hierarchical structures (~1 to 500 nm) in solution or in situ. Ultra-SANS extends accessible length scales to several microns. Time-resolved SANS experiments are also possible for kinetic studies with timescales typically longer than those for SAXS (seconds to minutes).

Solution X-Ray Scattering

Solution X-Ray Scattering is also known as Small-Angle X-Ray Scattering. SAXS is a versatile technique that interrogates non-crystalline biological materials including solutions and gels. X-rays scattered from these materials can be analyzed to extract a wide array of structural, dynamic, and temporal properties. Often biological macromolecules can be solubilized in a homogeneous form with identical copies of the same assembly throughout the solution. In such cases, SAXS can be performed in high throughput (HT-SAXS) to assess how solution conditions, co-factors, or small modifications affect conformation and assembly. Hundreds of conditions can be tested in a few hours. In other cases, assemblies are transient and can only be isolated into homogeneous particles during purification. SAXS data can be collected during purification with the elution from a size exclusion purification column (SEC-SAXS) flowing directly in front of the X-ray beam. SAXS can assess conformational changes as small as 5 Å and can be collected at submillisecond timescales. SAXS has been used in hundreds of structural studies of proteins, virus particles, and biological fibers as well as lipid membranes and membrane protein–DNA complexes.

X-Ray Macromolecular Crystallography

This is a widely used technique based on diffraction from crystalline biological materials [including proteins, large protein complexes, and nucleic acids (RNA and DNA)] to obtain high-resolution structural information (often in the ~1–2 Å range). MC requires that the biomolecules be crystallized, but can provide specific atom locations in very complex systems, enabling detailed insight into how these macromolecules carry out their functions in living cells and organisms.