A resolution of 0.3 nm has been achieved by electron microscopy—enough to begin to see the internal atomic arrangements in a protein and to rival xray crystallography in resolution. Although electron microscopy is unlikely to supersede xray crystallography (discussed in Chapter 8) as a method for macromolecular structure determination, it has some very clear advantages. First, it does not absolutely require crystalline specimens. Second, it can deal with extremely large complexes—structures that may be too large or too variable to crystallize satisfactorily.  Third, it allows the rapid analysis of different conformations of protein complexes.
The analysis of large and complex macromolecular structures is helped considerably if the atomic structure of one or more of the subunits is known, for example from xray crystallography. Molecular models can then be mathematically “fitted” into the envelope of the structure determined at lower resolution using the electron microscope (see Figures 16–16D and 16–46). Figure 9–55 shows the structure of a ribosome with the location of a bound release factor displayed in this way (see also Figure 6–72).
Discovering the detailed structure of membranes and organelles requires the higher resolution attainable in a transmission electron microscope. Specific macromolecules can be localized after being labeled with colloidal gold linked to antibodies. Three-dimensional views of the surfaces of cells and tissues are obtained by scanning electron microscopy. The shapes of isolated molecules can be readily determined by electron microscopy techniques involving fast freezing or negative staining. Electron tomography and single-particle reconstruction use computational manipulations of data obtained from multiple images and multiple viewing angles to produce detailed reconstructions of macromolecules and molecular complexes. The resolution obtained with these methods means that atomic structures of individual
Figure 9–54 Single-particle reconstruction. the structure of a complete human immunodeficiency virus (hiV) capsid has been determined by a combination of cryoelectron microscopy, protein structure determination, and modeling. (a) a single 4 nm slice from an eM tomographic model (see also Figure 9–48) of an intact hiV particle with its membrane outer envelope and its internal, irregularly shaped protein capsid that houses its rna genome. (B) electron microscopy of capsid subunits that have self-assembled into a helical tube can be used to derive an electron-density map at a resolution of 8 nm, in which details of the hexamers are clearly visible. (C) Using the known atomic coordinates of a single subunit of the hexamer, the structure has been modeled into the electron-density map from (B). (D) a molecular reconstruction of the entire hiV capsid, based on the detailed structures shown in (a) and (C). this capsid contains 216 hexamers (blue) and 12 pentamers (yellow). (adapted from g. Zhao et al., Nature 497:643–646, 2013. With permission from Macmillan publishers Ltd. C, pDB code: 3J34.)
Figure 9–55 Single-particle reconstruction and molecular model fitting. Bacterial ribosomes, with and without the release factor required for peptide release from the ribosome, were used to derive high-resolution, three-dimensional cryoelectron microscopy maps at a resolution of better than 1 nm. images of nearly 20,000 separate ribosomes preserved in ice were used to produce single-particle reconstructions.