Student Seminar: Cryo–Electron Microscopy as a Structural Biology Tool: Principles, Methodology, and Applications

High-resolution structural characterization of biological macromolecules is central to understanding the molecular mechanisms that govern cellular function.¹ Although X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have provided most atomic models to date, these techniques face inherent limitations when applied to large, heterogeneous, conformationally dynamic, or membrane-embedded complexes. Cryo-electron microscopy (cryo-EM) has emerged as a powerful alternative, enabling structure determination of macromolecular assemblies in a near-native, non-crystalline state without the requirement for long-range order. Developments in the electron microscopy of frozen hydrated samples (cryo – electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules.

The Royal Swedish Academy of Sciences has decided to award Jacques Dubochet, Joachim Frank and Richard Henderson the Nobel Prize for Chemistry 2017 for “developing cry electron microscopy for the high-resolution structure determination of biomolecules in solution”. The year 1990 marked a critical milestone when Henderson and colleagues showed for the first time that it is possible to obtain high-resolution structures of biomolecules using cryo-EM through averaging over many copies of the same object.^2,3A fundamental problem in studies of unstained, non-crystalline, asymmetrical, randomly oriented particles in solution is “the alignment of features that are only faintly visible on a noisy background”. In the mid-1970s, Frank addressed this problem in a study that became in many ways the starting point for future developments.⁴ Sample preparation is a critical determinant of structural resolution and data quality in cryo–electron microscopy (cryo-EM). In this context, the pioneering contributions of Jacques Dubochet were transformative. In 1984, Dubochet and colleagues demonstrated the full potential of rapid vitrification by successfully imaging virus suspensions embedded in thin, unsupported films of vitreous ice.⁵ This methodological breakthrough prevented ice crystal formation and preserved biological specimens in a near-native hydrated state, thereby enabling high-resolution structural analysis.^6,7

This seminar will provide an overview of cryo–electron microscopy (cryo-EM), with particular emphasis on its instrumentation, advanced sample preparation strategies, and its role in the high-resolution structural determination of proteins.