Electron Cryomicroscopy and Three-dimensional Computer Reconstruction of Biological Molecules

The transmission electron microscope (TEM) is a versatile device used in fields ranging from material science to cell biology to biochemistry. In a light microscope, the light wavelength is typically about 5000 Å, which makes it the primary resolution-limiting factor. In aTEM,however, the electron wavelength is typically less than 0.04 Å, much smaller than the size of a single atom. The resolutionlimiting factors in TEMs vary with the type of specimen being examined, but for biological molecules and macromolecules in the 100–10 000-Å range, radiation damage is the primary limiting factor. Doses of less than 15 electrons per Å are generally sufficient to destroy structures at near atomic resolution, even when the specimen is kept at a temperature below 2 1608C. An additional difficulty lies in the requirement that the specimen chamber be maintained under high vacuum. Unlike the photons employed in light microscopes and Xray diffraction experiments, electrons cannot travel coherently through the air. Clearly this makes it impossible to use liquid specimens in a TEM. To avoid this problem, the specimen is fixed in a thin layer of metallic stain, glucose or vitreous ice. Vitreous ice provides the best specimen preservation at high resolution. In this technique, a thin layer of the macromolecules in solution is prepared on a microscope grid, then frozen very rapidly by plunging into liquid ethane. This freezing process is so rapid that it produces a layer of vitreous ice nearly identical in structure to liquid water. The molecule is maintained in a welldefined chemical environment and conformation by continuously holding it below 2 1608C during measurement. Ice sublimation in the microscope vacuum is negligible. Images collected by means of a TEM suffer from an artefact known as the contrast transfer function, caused by the electron optics (Erickson andKlug, 1970). Images may also suffer from astigmatism, charging, drift and other problems. Effects such as beam coherence and sample holder stability also limit the resolution that can be obtained in a given microscope. Correction of these artefacts and the evaluation of data quality are nontrivial problems thatmust be addressedwhen performing a threedimensional (3D) reconstruction.