Development of High-Resolution Transmission Electron Microscopes for Analysis of Biomolecular Structure

Abstract

Determining the structure of biological macromolecules, such as proteins and nucleic acids, is important for understanding their function. This leads to a mechanistic understanding of biological systems, and suggests pathways for discovery of therapeutic drugs. Structural analysis of biological macromolecules was first demonstrated in 1968 by DeRosier and Klug, with their three-dimensional reconstruction of negatively stained bacteriophage T4 [1]. Since liquid water is not compatible with the vacuum of an electron microscope, yet water retention is essential to maintain high-resolution molecular structure, methods of freezing the water in a vitreous state were eventually developed. This enabled the molecules to be imaged in a near-native state in the electron microscope. Yet, the high-resolution structure of these samples is easily compromised by electron-beam irradiation. As expected, lowering the temperature of the sample in the electron microscope was shown to reduce radiation sensitivity, and the lowest temperatures provided the most protection [2]. Thus, an electron microscope with a cryostage to maintain the low temperature, and a cryo-transfer system to avoid frosting or devitrification during specimen insertion, was called for.In 1983, development of a dedicated cryo-transmission electron microscope (cryo-TEM) was started jointly by Y. Fujiyoshi and JEOL. Membrane proteins were chosen as the first specimens to be imaged, since they play important functions in the cell for trans-membrane transport and signaling, and knowledge of the structure and function of membrane proteins at the molecular level may clarify disease mechanisms. The three-dimensional structure of macromolecules can be obtained by several methods, including electron tomography, single-particle analysis, and electron diffraction. We chose electron diffraction, optimized for cryo-TEM. Either x-ray or electron diffraction can yield atomic or nearatomic results. Previously, x-ray diffraction was favored for obtaining the best resolution, but a threedimensional crystal is required. Electron diffraction is carried out with two-dimensional crystals, which are often easier to produce from biological macromolecules. By embedding two-dimensional crystals in lipid layers, followed by vitreous freezing, we were able to use electron diffraction to study membrane proteins at near-atomic resolution in a near-native state.We used a liquid-helium reservoir centrally located in the TEM column to cool the specimen stage. The helium reservoir was surrounded by a liquid-nitrogen tank to suppress thermal radiation from the surrounding room environment. In order to avoid vibration from bubbling of the liquid helium, we made use of super-fluidity to cool the specimen, by which we could reach a temperature close to 2K. In keeping with best practices at the time, we employed a top-entry stage design for optimum stability. After much trial-and-error, the first-generation cryo-TEM was completed in 1986, which enabled highresolution imaging at 4.2K. Since then, the c...