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Review
. 2015 May 21;58(4):677-89.
doi: 10.1016/j.molcel.2015.02.019.

Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity

Eva Nogales  1 , Sjors H W Scheres  2
Affiliations
Review

Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity

Eva Nogales et al. Mol Cell. .

Abstract

3D cryo-electron microscopy (cryo-EM) is an expanding structural biology technique that has recently undergone a quantum leap progression in its achievable resolution and its applicability to the study of challenging biological systems. Because crystallization is not required, only small amounts of sample are needed, and because images can be classified in a computer, the technique has the potential to deal with compositional and conformational mixtures. Therefore, cryo-EM can be used to investigate complete and fully functional macromolecular complexes in different functional states, providing a richness of biological insight. In this review, we underlie some of the principles behind the cryo-EM methodology of single particle analysis and discuss some recent results of its application to challenging systems of paramount biological importance. We place special emphasis on new methodological developments that are leading to an explosion of new studies, many of which are reaching resolutions that could only be dreamed of just a couple of years ago.

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Figures

Figure 1
Figure 1. Basic concepts of cryo-EM structure determination
(A) The projection-slice theorem states that the 2D projection of a 3D object in real-space (left column) is equivalent to taking a central 2D slice out of the 3D Fourier transform of that object (right column). The realspace projection direction (left; dashed red arrows) is perpendicular to the slice (right; red frame). (B–E) Many experimental 2D projections can be combined in a 3D reconstruction through an iterative process called “projection matching”. To determine the relative orientations of all experimental projections one first calculates reference projections of a 3D object in all directions (B). Then, one compares each experimental projection with all reference projections to find the best match of a given similarity measure (C). This orients all experimental projections relative to the 3D structure (D). The projection-slice theorem then implies that the 3D reconstruction can be calculated by positioning many 2D slices (the 2D Fourier transforms of all experimental projections) into the 3D transform (E) and calculating an inverse transform. Iterating steps (B–E) will gradually improve the orientations, and hence the resolution of the reconstruction.
Figure 2
Figure 2. Study of the human transcription PIC
(A) Negative stain reconstructions of increasingly larger human PIC assemblies. (B) Detail of a PIC cryo-EM reconstruction showing Pol II contacts with duplex DNA in the closed PIC complex. (C) Comparison of the closed and open PIC cryo-EM structures. (D) Negative stain reconstruction of the TFIIH-containing PIC with available atomic models. Modified from He et al. (2013) Nature.
Figure 3
Figure 3. High-resolution cryo-EM structures of dynamic and stabilized microtubules
(A) Cryo-EM map of the GMPCPP MT (4.7 Å resolution). α-tubulin, green, β-tubulin, blue. (B) β- tubulin C-terminal helices (left) and beta strands in the α-tubulin intermediate domain (right) from the Rosetta GMPCPP MT model. (C) Cα traces of two longitudinally associated tubulin dimers from the GMPCPP (gold) and GDP (light purple) Rosetta models, superimposed on the underlined β tubulin, showing that hydrolysis results in a compression of the E-site at the interdimer interface (box). View is tangential to the microtubule lumen. The cartoon of a MT GTP cap illustrates how the two states described could coexists at a MT end. (D) Interdimer interface by the E-site nucleotide showing the conformational changes from GMPCPP (semitransparent) to GDP (solid).
Figure 4
Figure 4. High-resolution cryo-EM structures from heterogeneous samples
(A) To some extent, the classical approach in structural biology to study biochemically purified, homogeneous samples may be bypassed by cryo-EM image processing, where images of a mixture may be separated in the computer using powerful classification algorithms to obtain high-resolution structures for multiple components in the mixture. (B) Provided enough particles may be identified for each component, atomic-resolution maps may be generated for each of them. (C) Even small-molecule compounds may be built inside the high-resolution maps, in this case the eukaryotic translation inhibitor emetine is shown bound to the cytoplasmic ribosome from the P. falciparum parasite.
Figure 5
Figure 5. Near-atomic resolution cryo-EM structure of human gamma-secretase
(A) Overall view of the complex with the trans-membrane domain (TMD), which is made up of the four different proteins, in blue, and the extra-cellular domain (ECD) of Nicastrin in green. (B) Representative density for the soluble domain showing a region of the map with separated betastrands. (C) View inside the TMD perpendicular to the membrane showing the horse-shoe like arrangement of the trans-membrane helices with a thick and a thin end. The lack of good sidechain density in this region of the map prohibited the assignment of each helix to the four different proteins.
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