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

Techniques Overview

Structural Biology Techniques and Length Scales

Structural Biology Techniques and Length Scales. Critical structures and functions in biology occur across a wide range of distances (subnanometer to centimeter) and times (subpicosecond to minutes). Today, advanced imaging and characterization techniques enable researchers to image molecules, complex biological machines, native cellular structures, and tissue architectures at or near atomic-level resolution and with high temporal resolution. Such techniques are available to the research community through BSSD-supported structural biology resources at DOE user facilities across the country. [See bottom of page for image credits.]

Today’s advanced imaging and characterization techniques enable researchers to image molecules, complex biological machines, native cellular structures, and tissue architectures at or near atomic-level resolution and with high temporal resolution. These techniques, including X-ray, neutron, and cryo-electron microscopy capabilities, are freely available to the scientific community at U.S. Department of Energy (DOE) user facilities funded by the Office of Basic Energy Sciences (BES) and the Office of Biological and Environmental Research (BER).

At these facilities, BER supports beamlines and resources to enable multiscale structural studies. New insights linking molecular properties to system-level functions can be achieved by integrating these different capabilities to make cross-scale connections and leveraging data from complementary techniques such as super-resolution optical and magnetic resonance imaging.

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 Imaging

Neutron imaging includes neutron radiography and computed tomography. Taking advantage of H/D contrast, and the high penetrating power and the nondestructive nature of neutrons, it is possible to study structures of a wide range of hierarchical and complex materials of biological relevance at a resolution of ~50 µm. Examples include transport and interactions of fluids in porous media, plant-plant and plant-fungal interactions, pore structure and voids in soil under environmentally relevant conditions, and cavitation and gas embolism in plant-soil-groundwater systems.

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.

Neutron Spectroscopy

This is a two-dimensional (2D) technique that provides information about atomic motions in both time and space. Inelastic and quasi-elastic neutron scattering provide information about vibrational modes, molecular motions, and diffusive properties of biomolecules and their hydration water on the picosecond to nanosecond timescale. Neutron spin echo spectroscopy probes slower motions at microseconds to milliseconds, such as motions associated with undulating membranes and domain motions in proteins.

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).

Soft X-Ray Tomography

There are several variants of X-ray imaging, including 2D and 3D (tomography), that use both soft and hard X-rays. Soft X-ray tomography can be used to image the 3D structural organization of whole, hydrated biological cells in the native state down to a resolution of about 35 nm. Hard X-ray tomography can provide 2D and 3D information on more strongly absorbing and less radiation sensitive biological materials with a resolution of 30 nm or less. A variant of X-ray imaging, called spectromicroscopy, provides spatially resolved information about metal distribution and chemical speciation in materials of biological and medical relevance, including tissues with resolutions typically from submicrons to millimeters.

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.

Synchrotron Infrared Hyperspectral Imaging

Unlike X-rays, noninvasive infrared photons carry energy quanta matching closely with molecular structure–specific vibrational energy levels. These molecular vibrational energy levels can be subtly perturbed by changes in the local chemical environment. Since this specific intrinsic contrast mechanism removes the requirement for specific tags commonly used in traditional optical imaging technologies, infrared absorption spectroscopy is ideally suited for exploratory analyses of the chemistry and structure of biological materials, cells, and tissues with a spatial resolution of 10 nm to 15 µm. Time-resolved infrared spectroscopy can be used to study chemical and structural changes in living biological systems, with timescales ranging from seconds to minutes, and to identify prospective regions and time-points of interest.

X-Ray Absorption and Emission Spectroscopy

Metal ions have key roles in biological structure and function. For example, they are the active sites of many enzymes and shuttle electrons in key metabolic or signaling pathways. This suite of related techniques provides both structural and electronic information on metal sites in biomolecules. X-ray absorption edge spectroscopy can reveal oxidation state and chemical states of elements in complex environments. Extended X-ray absorption fine structure (EXAFS) can determine high-resolution bond distances, revealing the local structure around a metal ion. Related techniques such as X-ray emission, resonant inelastic scattering, and X-ray Raman can provide a detailed picture of metal-ligand bonding. XAS methods provide very complementary information to data from X-ray crystallography and small-angle scattering studies. This technique also is applied to studies of biologically important ligands (e.g., carbon, nitrogen, sulfur, and chlorine) and their interactions with metals.

X-ray Fluorescence Imaging

X-ray fluorescence imaging (XRF), or x-ray spectromicroscopy, maps the distributions of elements and chemical species of interest within biological samples. Synchrotron XRF (SXRF) can provide detailed images of element speciation to a resolution of 0.5 µm per pixel, a sensitivity beyond desktop XRF, electron microprobe, or other elemental imaging techniques.

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.

Structural Biology Techniques and Length Scales: Courtesy Stanford Synchrotron Radiation Lightsource at SLAC National Accelerator Laboratory. Individual images left to right:  (1) Van Stappen, C., et al. 2019. “Spectroscopic Description of the E1 State of Mo Nitrogenase Based on Mo and Fe X-Ray Absorption and Mössbauer Studies,” Inorganic Chemistry 58(18), 12365–376. DOI:10.1021/acs.inorgchem.9b01951. Reprinted under a Creative Commons License (CC BY 4.0). (2) Kim, Y., et al. 2021. “Tipiracil Binds to Uridine Site and Inhibits Nsp15 Endoribonuclease NendoU from SARS-CoV-2,” Communications Biology 4, 193. DOI:10.1038/s42003-021-01735-9. Reprinted under a Creative Commons License (CC BY 4.0). (3) PDB ID: 6MOR. Roh, S. H., et al. 2020. “Cryo-EM and MD Infer Water-Mediated Proton Transport and Autoinhibition Mechanisms of Vo Complex,” Science Advances 6(41), eabb9605. DOI:10.1126/sciadv.abb9605. (4) Courtesy Thomas SpleIstoesser, See also Vandavasi, V. G., et al. 2016. “A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers,” Plant Physiology 170(1), 123–35. DOI:0.1104/pp.15.01356. (5) Reprinted with permission from Roth, M. S., et al. 2017. “Chromosome-Level Genome Assembly and Transcriptome of the Green Alga Chromochloris zofingiensis Illuminates Astaxanthin Production,” Proceedings of the National Academy of Sciences USA 114(21), E4296–4305. DOI:10.1073/pnas.1619928114. (6) Martin, M. C., et al. 2013. “3D Spectral Imaging with Synchrotron Fourier Transform Infrared Spectro-Microtomography,” Nature Methods 10, 861–64. DOI:10.1038/nmeth.2596. (7) Seyfferth, A. L, et al. 2017. “Evidence for the Root-Uptake of Arsenite at Lateral Root Junctions and Root Apices in Rice (Oryza sativa L.),” Soils 1(1), 3. DOI:10.3390/soils1010003. Reprinted under a Creative Commons License (CC BY 4.0). (8) Courtesy Oak Ridge National Laboratory. See also Dhiman, I., et al. 2017. “Quantifying Root Water Extraction After Drought Recovery Using sub-mm In Situ Empirical Data,” Plant Soil 417, 1–17. DOI:10.1007/s11104-017-3408-5.