The ability to understand life at its most fundamental level rests on knowing the precise shape of biological molecules, often summarized as structure determining function. Cryogenic Electron Microscopy (Cryo-EM) is a modern scientific technique that allows researchers to visualize these molecular machines at near-atomic resolution. By rapidly freezing purified samples, Cryo-EM captures a molecule’s three-dimensional structure, providing the blueprint necessary to understand how it performs its job within a cell. The technology has revolutionized structural biology, offering clarity previously unattainable with older methods.
Limitations of Traditional Structure Determination
Before Cryo-EM, scientists primarily relied on X-ray crystallography to determine the shape of proteins, a method that requires the molecules to be forced into a rigid, repeating crystal lattice. This crystallization step often failed for complex, dynamic biological machines or for molecules that naturally reside in cell membranes, such as receptors and channels. Membrane proteins are particularly challenging, yet they are the target of nearly two-thirds of all pharmaceutical drugs. The static nature of a crystal structure also fails to capture the multiple flexible shapes a protein assumes during its working life.
Another traditional imaging method, room-temperature electron microscopy, suffered from an entirely different problem: the high-energy electron beam necessary for imaging would instantly destroy the delicate biological sample. This severe radiation damage caused mass loss and bond breakage. The biological material would collapse in the microscope’s vacuum environment, rendering the resulting images useless for accurate structural analysis. These limitations established a clear need for a method that could image molecules in a more natural state without damaging them.
The Key to Cryo-EM: Vitrification
The breakthrough that unlocked the potential of electron microscopy for biology was a specialized sample preparation technique called vitrification, which gives the method its “cryo” prefix. Vitrification is a process of ultra-rapid, flash-freezing the sample, typically by plunging a grid containing the suspended molecules into a liquid cryogen like ethane, cooled below -180°C. This extreme speed prevents water molecules from organizing into damaging ice crystals.
Instead of crystalline ice, the water forms a glass-like, non-crystalline solid known as amorphous or vitreous ice. This glassy state preserves the biological molecule in its native, hydrated environment and prevents it from collapsing in the microscope’s vacuum. The cryogenic temperature also significantly reduces the rate of radiation damage caused by the electron beam. Vitrification is the physical step that makes high-resolution imaging possible.
From 2D Images to 3D Structures
Once vitrified, the sample is transferred into an electron microscope, where it is subjected to an extremely low-dose electron beam to minimize radiation damage. The electrons pass through the thin layer of ice and the embedded molecules, generating thousands of faint, low-contrast, two-dimensional projection images known as micrographs. Because the molecules are randomly oriented on the grid, each 2D image represents the molecule from a different viewing angle.
The subsequent process, called single-particle reconstruction, is largely a computational feat. Specialized software algorithms first identify the thousands of individual molecular images, or “particles,” in the noisy micrographs. These particles are then sorted into groups based on their orientation, aligned, and computationally averaged. This averaging step boosts the signal-to-noise ratio, transforming the initially fuzzy, low-contrast data into a sharp, high-resolution three-dimensional density map. This final map reveals the location of the atoms, providing the definitive molecular structure.
Visualizing Biological Machinery
The ability of Cryo-EM to image molecules in their native state has provided unprecedented insights into the largest and most dynamic components of the cell. For example, Cryo-EM has successfully solved the near-atomic structures of the ribosome, the complex responsible for protein synthesis, revealing the precise mechanism by which it translates genetic code. It has also mapped the spliceosome, a massive molecular machine that edits RNA, capturing its complex movements and multiple intermediate states during the splicing reaction.
The technique has proven particularly powerful in virology, where the structures of complex viral capsids were quickly determined during public health crises. Within weeks of the COVID-19 pandemic’s onset, Cryo-EM provided the structure of the SARS-CoV-2 spike protein, the molecule the virus uses to enter human cells. This structure was resolved at 3.5 Å resolution, immediately revealing its shape and providing a molecular blueprint for vaccine and therapeutic development.
The Revolution in Drug Discovery
The structural information provided by Cryo-EM has fundamentally altered the landscape of drug discovery, enabling a more precise, structure-based approach to medicine. By visualizing a target protein in its natural conformation, scientists can precisely map the three-dimensional contours of its active sites and binding pockets. This level of detail allows researchers to design small-molecule drugs that fit perfectly, optimizing the drug’s effectiveness and minimizing off-target effects.
Cryo-EM has been used to determine the binding modes of existing antibiotics to the ribosome and to discover new binding sites on challenging drug targets, such as G-protein-coupled receptors (GPCRs). The technique’s speed to handle previously intractable molecules accelerates the identification and modification of therapeutic compounds. The impact of the technique on structural biology was formally recognized in 2017 when the Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson for its development.