Cryo-EM vs. X-ray Crystallography
Cryo-electron microscopy (cryo-EM) and X-ray crystallography are two essential techniques for enhancing our understanding of biological macromolecules at atomic resolution. Though distinct in methodology and application, these techniques are highly complementary, with each filling gaps left by the other. As researchers go deeper into the complex mechanisms underlying biological processes, leveraging both methods offers unparalleled insights, pushing structural biology toward new heights of discovery.
X-ray Crystallography: The Gold Standard
Historically, X-ray crystallography has been the predominant technique in structural biology. In this method, an X-ray beam passes through a crystallized sample and a diffraction pattern is created when the X-rays interact with the well-ordered crystal lattice. By analyzing the Bragg reflections in these patterns in terms of amplitudes and phases researchers can reconstruct the molecule's three-dimensional structure typically with atomic precision. The sharpness of these diffraction spots is key, directly correlating to the quality and order of the crystal. However, completeness of the reflections in each resolution zone plays another essential role in determining the final resolution of the three-dimensional structure and thus to what extent the structure can be interpreted.
However, a notable challenge is that not all biomolecules are easy to crystallize. Many biologically significant structures, such as membrane proteins or large, flexible macromolecular complexes, resist crystallization due to their dynamic nature. Also, crystallization often requires significant sample quantities and molecular engineering to stabilize flexible regions—limitations that have sparked the search for alternative methods. Furthermore, intermediary structures that can provide snapshots of important dynamic processes are extremely hard to crystallize.
Despite these hurdles, X-ray crystallography remains unsurpassed in resolving fine atomic details of well-ordered macromolecules. Its precision makes it the go-to technique for studying stable and crystallizable proteins, enzymes, and other biomolecules. Structural biologists use it to uncover mechanistic details that are crucial for drug development and understanding molecular interactions.
Cryo-Electron Microscopy: Flexibility for Non-Crystalline Samples
Cryo-EM, by contrast, doesn't require crystallization. Instead, biological samples are flash-frozen, capturing molecules in their near-native state. A high-energy electron beam is passed through these frozen samples, producing 2D projections of said molecules at various orientations. These images are then computationally assembled into a 3D map of the molecule.
Cryo-EM has surged in popularity, especially for large macromolecular complexes that are difficult or impossible to crystallize. Recent advancements in cryo-EM technology—such as improved electron detectors and image-processing algorithms—have led to what is referred to as the "resolution revolution." With cryo-EM, researchers can now achieve near-atomic resolution for even flexible or heterogeneous assemblies, such as viruses, ribosomes, and protein complexes.
One of cryo-EM's primary strengths is its ability to visualize large, multi-component structures in different conformational states. This flexibility enables scientists to capture dynamic interactions within macromolecular machines, something X-ray crystallography struggles to achieve. However, cryo-EM's resolution typically doesn't match the atomic-level precision of crystallography, particularly for smaller proteins or structures below 100 kDa.
Complementary Roles in Structural Biology
Despite their differences, cryo-EM and X-ray crystallography are not competing technologies but rather complementary tools that can work together for more complete structural insights. One of the most practical synergies between these methods is solving structures through an integrated approach.
For instance, cryo-EM can generate an initial low- to medium-resolution 3D map of a large protein complex, providing the overall architecture. X-ray crystallographic data of individual components can then be docked into the cryo-EM map, revealing high-resolution details of specific domains or subunits within the larger complex. This combined approach has been particularly valuable in studying systems that are too dynamic or heterogeneous for crystallography alone. Well before the so-called ‘resolution revolution’ this was effectively the go-to technique in the 1980s to elucidate large complexes at resolution beyond a nanometer.
Conversely, cryo-EM can assist X-ray crystallography by solving one of its most notorious hurdles—the phase problem. X-ray diffraction data capture only the intensity of the Bragg wave, but not its phase, making it impossible to resolve structures of large biomolecular complexes without a suitable means to derive these phases. Crystals of small compounds, such as paraffins, can often be solved using direct methods. These types of samples are also extremely well-suited to be solved using microED. Cryo-EM maps can provide an initial model for molecular replacement, thus enabling researchers to determine phases and solve the crystal structure at higher resolution.
Additionally, the distinct physical principles underlying the two methods—X-ray photons interacting with electron clouds in crystallography versus high-energy electrons interacting with atomic Coulomb potentials in cryo-EM—mean they provide slightly different structural information. In practice, this allows researchers to study molecules in different states or conformations, offering a more holistic view of biological structure and function.
Future Directions: Integration for Enhanced Discovery
As both cryo-EM and X-ray crystallography technologies continue to advance, the lines between them may blur further, with researchers increasingly using both techniques in tandem to solve complex structural puzzles. With cryo-EM achieving near-atomic resolution and micro-electron diffraction (microED) emerging as a method to solve crystallography-style biomolecules with very small crystals, structural biology is poised for breakthroughs that were unimaginable just a few decades ago.
JEOL has been at the forefront of this evolution, providing
state-of-the-art cryo-EM systems that offer unmatched resolution and flexibility. By integrating cryo-EM with X-ray crystallography data, researchers using JEOL instruments can push the boundaries of structural biology, revealing intricate biological systems in unprecedented detail.
Ultimately, leveraging both cryo-EM and X-ray crystallography allows scientists to investigate macromolecular structures with an accuracy and completeness that no single method can achieve alone. As structural biology progresses, the combination of these complementary techniques will continue to drive innovation and discovery across the life sciences.
References & Further Reading
- Wang HW, Wang JW. How cryo-electron microscopy and X-ray crystallography complement each other. Protein Sci. 2017 Jan;26(1):32-39. doi: 10.1002/pro.3022. Epub 2016 Sep 7. PMID: 27543495; PMCID: PMC5192981.