Electron Cryomicroscopy of Molecular Nanomachines and Cells

Abstract

Electron cryomicroscopy (cryo‐EM) is an emerging biophysical tool that can be used to determine the structures of molecular nanomachines in fully solvated conformations at subnanometer resolutions (<1 nm). Such cryo‐EM maps can reveal long α‐helices and large β‐sheets. In the highest resolution cryo‐EM density maps, it is possible to see bulky side chains and trace the Cα backbone of molecular subunits within a multicomponent nanomachine. Electron cryotomography (cryo‐ET) is equally powerful because of the unique cellular context in which it can capture and reveal cellular nanomachines. Despite reaching only 4–10 nm resolution, cryo‐ET reconstructions are capable of imaging whole cells and distinguishing their molecular components. Both of these methods are complementary to conventional methods of structure determination, including X‐ray crystallography and NMR spectroscopy. Hybrid methods that combine these structural techniques with cryo‐EM and cryo‐ET result in a complete view of nanomachines from atomic detail to their spatial and temporal location within a cell. This chapter will describe the experimental and computational pipeline in cryo‐EM and cryo‐ET and illustrate their effectiveness with examples from an ion channel, spherical virus, cellular motor and the survey of a complete cell.

Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus

“…Cryo-ET is an emerging method for reconstructing the 3-D structure of one sample at a time 42 , 43 . Cryo-ET is a low-resolution technique because of the radiation damage to specimens resulting from multiple electron exposures, but it is extremely powerful for revealing structures that may vary from one cell to another and therefore cannot be averaged as in a single particle reconstruction.…”

Zernike Phase Contrast Cryo-Electron Microscopy and Tomography for Structure Determination at Nanometer and Subnanometer Resolutions

“…In cryo-electron microscopy (cryoEM), biological specimens, such as purified proteins, virus particles, organelles and cells, are embedded in amorphous ice by rapid freezing (Dubochet et al, 1988; Baker et al, 2009), and directly observed in the microscope, maintaining them at low temperature. The frozen, hydrated specimens can be considered as phase objects in transmission electron microscopy, and the 3-D density function of their contents can be retrieved from the images (Toyoshima and Unwin, 1988).…”