Introduction

It is difficult nowadays to provide an introduction into cryo-EM within the space of a book chapter, given the current plethora of different methods, and the fact that there are as yet no agreed-on standards in the field. In view of this situation, the best course for the author is to provide the reader with an illustrated introduction into important concepts and strategies. However, at the same time, the focus on the molecular machine invites an expansion of scope in the most relevant section (Section IV), which concerns itself with heterogeneity, and the challenge to obtain an inventory of conformational states of a molecular machine in a single scoop.

Preliminaries: Cryo-EM as a Technique of Visualization

The transmission electron microscope (TEM) produces images that are projections of a three-dimensional object. To be more precise, the projections are line integrals of the three-dimensional Coulomb potential distribution representing the object. (For all practical purposes, especially in the resolution range down to to ∼3 Å, the Coulomb potential distribution is identical to the electron density distribution “seen” by X-rays). Visualizing a molecular machine in three dimensions therefore entails the collection of multiple images showing the molecule in the same processing state (and hence, identical structure) but in different views. Thus the term “3D electron microscopy” is understood as a combination of two-dimensional imaging, following a particular data collection strategy, with three-dimensional reconstruction.