X-ray Macromolecular Crystallography

Atomic-resolution images of proteins, RNA, DNA, complexes, and macromolecular machines

New insight into overcoming plant recalcitrance

Carboxylases are key enzymes in photosynthesis and the global carbon cycle. The structure of enoyl-CoA carboxylase/reductase from a soil bacterium sheds light on the molecular basis of one of nature’s fastest CO2-fixing enzymes. X-ray crystallography of the tetramer structure (four colored protein subunits), combined with molecular dynamics calculations, revealed the CO2-fixing mechanism and interdomain interactions that enable remarkably fast turnover of the CO2-fixing reaction. This paves the way for designing efficient and selective CO2-fixing enzymes for biofuel and biomolecule production. [From Stoffel, G. M. M., et al. 2019.]

X-ray macromolecular crystallography (MX) uses X-rays to determine the atomic-level structures of biological molecules across a broad range of sizes and complexity. In this widely used technique, high-intensity X-rays are directed onto crystals containing biological molecules to obtain a series of diffraction images that are then assembled into a dataset. The dataset is analyzed to yield high-resolution structural information (often in the ~1–3 ångström resolution range). This provides the precise location of the often thousands of atoms in the structure. Such information can be obtained for a wide range of biological systems, including soluble and membrane-bound proteins, enzymes, nucleic acids (RNA and DNA), glycans, other post-translational protein modifications, and complexes of these molecules. MX can determine the structures of complex macromolecular systems and even whole viruses and can investigate bound metabolites or potential drugs, metal ions, and other cofactors.

Such knowledge provides detailed insight into how these biological systems function in living cells, tissues, and organisms. Detailed structural information is crucial for identifying the active and regulatory sites of enzymes participating in key metabolic or signaling pathways in soil microorganisms and plants, and is essential to structure-based drug discovery. MX structural information about engineered and de novo designed enzymes can also provide crucial information for synthetic biology research.

Most MX studies today are conducted using synchrotron or free-electron laser X-ray sources. These sources generate extremely bright X-ray beams producing the highest quality structural information, provide automation for very high throughput sample analysis, and offer remote access and expert support. MX-acquired information is highly complementary to neutron diffraction, cryo-EM imaging, solution X-ray scattering, neutron scattering, nuclear magnetic resonance spectroscopy, and functional studies.

Envelope structure and scattering properties of <em>B. subtilis</em>

X-ray crystallography provides structural insight into how SARS-CoV-2 interacts with human cells. Here, the virus’ receptor-binding domain (RBD, gray), located on the spike protein, binds to ACE2 (green), the receptor on human cells which enables entry of the virus into a cell. Such structural details inform strategies to engineer antibodies that prevent infection with SARS-CoV-2, the virus that causes COVID-19.  [Reprinted under a Creative Commons Attribution 4.0 International (CC BY 4.0) license from Kyere, J., et. al. 2020. "A Therapeutic Non-self-reactive SARS-CoV-2 Antibody Protects from Lung Pathology in a COVID-19 Hamster Model," Cell 183(4), 1058-1069, e19. DOI: 10.1016/j.cell.2020.09.049.]

Key Features of X-ray Macromolecular Crystallography

  • Accommodates biological molecules ranging in size from tens of daltons to hundreds of kilodaltons, including large macromolecular complexes and whole viruses.
  • New MX technologies enable sample measurements at physiological temperatures to monitor progress of chemical and structural transformations, such as during enzymatic catalysis.
  • Suites of data analysis software enable structure determination during and after data acquisition.
  • High-resolution crystal structures provide a strong basis for computational work on enzyme inhibitor/activator design and modeling of extremely large complexes such as fatty acid synthases, photosynthetic complexes, and cellulose synthase.

BER Researchers Use X-ray Macromolecular Crystallography to Study:

  • Molecules involved in biologically important processes such as biopolymer synthesis and degradation (e.g., glycosyl transferases, cellulases, and cellulosomes), energy conversion, and carbon cycling
  • Glycosidases in newly discovered microorganisms from the rhizosphere, hot springs (i.e., extremophiles), and animal rumens
  • Enzymes involved in carbon dioxide or nitrogen fixation and novel, engineered carbon and nitrogen cycling systems
  • Key enzymes in synthetic biology (e.g., CRISPR and engineered proteins)
  • Natural and engineered enzymes involved in biofuel production, such as polyketide synthases, terpene synthases, and P450s
  • Bacterial microcompartments that can sequester chemical reactions in a cell

See more examples in Science Highlights

Sample Considerations

  • Biomolecules must be purified in milligram quantities.
  • Samples must be crystallized in a form suitable for high-resolution data collection.
  • Typical high-resolution data collection requires cryogenic cooling of the crystals.
  • Low radiation dose data can be measured from crystals at room temperature when using serial crystallography at synchrotrons and X-ray free-electron lasers (XFELs).
  • XFELs enable structure determination of sensitive systems without radiation damage.
  • Data collection at ambient temperatures (2–25 oC) is possible.
  • Synchrotrons offer time-resolved crystallography data collection on millisecond to second timescales.
  • XFELs deliver femtosecond X-ray pulses that enable time-resolved measurements to image photochemistry and very fast reactions.

X-ray Macromolecular Crystallography Instruments 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.

  • 7 beamlines available for crystallographic studies of macromoleculesAdvanced Light Source
    Five beamlines are wavelength-tunable and optimized for multi-wavelength anomalous experiments. Several beamlines are equipped with high-speed, Pilatus pixel array X-ray detectors. All beamlines have automated sample-changing robots to facilitate rapid screening of crystals. Sample screening automation is combined with data collection software to enable remote, automated, unattended data collection. A new microfocus beamline, GEMINI, will begin operation in late 2021.
  • Structural Biology Center — Advanced Photon Source
    Provides access to two MX beamlines covering a wide energy range (6 to 20 keV). One beamline offers a microbeam for micrometer-size single crystals. Room temperature serial crystallography is available. Both beamlines are fully automated with remote access and robotic sample handling, and the center offers full user experiment control and access to its computing and software resources.
  • 5 beamlines — Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC
    2 beamlines — Linac Coherent Light Source (LCLS) at SLAC
    SSRL beamlines are equipped for MX studies, including two high-brightness microbeam undulator sources. All are fully automated and remotely accessible and controllable. Single-crystal ultraviolet–visible (UV-Vis) microspectrometry is available to monitor the electronic state of metalloenzyme crystals. Remote-accessible LCLS beamlines are equipped for MX studies as well. At both SSRL and LCLS, experimental capabilities for serial MX include fixed-target approaches (at cryogenic and elevated temperatures) and liquid/crystal injection-based approaches. SLAC also offers both complementary X-ray techniques (biological small-angle X-ray scattering and biological X-ray absorption spectroscopy imaging) and electron microscopy techniques (cryo-EM, cryo-ET, and cryo-focused ion beam scanning electron microscopy), with the opportunity for coordinated access to multiple instruments.

Citations

Stoffel, G. M. M., et al. 2019. “Four Amino Acids Define the CO2 Binding Pocket of Enoyl-CoA Carboxylases/Reductases,” Proceedings of the National Academy of Sciences USA 16(28), 13964–969. DOI: 10.1073/pnas.1901471116.