In particularly favorable cases, atomic-resolution structures are available for most, if not all, of the constitutive components of the assembly being studied by cryoEM. In such cases, subnanometer resolution EM maps not only permit delineation of subunit and domain boundaries, but discernable secondary structure elements allow for unambiguous positioning of atomic structures with an accuracy that far exceeds the resolution of the reconstruction itself. The resulting pseudo-atomic model of the entire macromolecular complex defines the precise location of functional elements, informs on protein-protein interfaces, and provides unique functional information about the complex as a whole.
Due to less stringent experimental requirements, it is common for low-resolution cryoEM reconstructions of macromolecular complexes to be solved before all (or even any) atomic-resolution structures of its assembly components are available. In these cases, the EM density may still provide valuable information about the arrangement of proteins within the complex. Docking of the available atomic structures into the cryoEM density can itself shed light on the position and role of the other portions of the complex. This is exemplified in the recent work of Lau and Rubinstein on the Thermus thermophilus
ATP synthase [14
]. ATP synthases consist of a membrane-embedded region, so far intractable by crystallography, that is connected to an extramembranous catalytic subcomplex by both a central stalk and one or more peripheral stalks. Lau and Rubinstein’s single particle analysis by cryoEM revealed the subnanometer structure of an intact ATP synthase, and docking of crystal structures provided key insights into the protein interactions within the catalytic cytoplasmic domain, as well as how these components are structurally coupled to the membrane-embedded ring subcomplex [16
Of particular importance, however, was the ability to define the remaining elements in the structure as the transmembrane helices of both the membrane-bound rotary ring and subunit I, revealing their mode of interaction. Two distinct clusters of helices within subunit I each interact exclusively with specific rotary subunits, one closer to the periplasm and the other closer to the cytoplasm (). This organization supports a two half-channel ion-translocating mechanism, in which one helical bundle of subunit I channels protons from the periplasm to the rotary subunits, while the other conducts protons from the rotary subunits to the cytoplasm. The authors propose that the movements of the rotary subunits are transferred to the central rotor by a funnel-shaped connector, linking the transmembrane proton motive force to ATP synthesis by the catalytic domains.
Figure 1 Organization of the ATP synthase and a model for proton translocation through the membrane. The crystal structures corresponding to the subunits in the extracellular domain are docked into the subnanometer EM reconstruction (EMDB ID: 5335)  to show (more ...)
The 26S proteasome is a classic example of a large macromolecular complex that has been the target of structural studies for several decades, but whose atomic structure remains elusive. The dynamic nature and labile character of the 19S regulatory particle (RP), which controls access to the proteolytic chamber, has significantly hampered efforts to define its structural organization. Atomic-resolution structures of some subunits or fragments had been determined [19
], but their relative arrangement within the RP could not be decisively defined, limiting our understanding of their contributions to RP function.
Two recent studies have combined cryoEM reconstructions of the 26S proteasome with biochemical data to provide a much more complete understanding of the RP architecture [22
]. Although making use of differing methodologies, these studies point to identical models of molecular organization. In the study by Lasker et al., cross-linking/MS experiments [24
] were combined with previously determined protein-protein interactions [25
] and crystal structures, and placed in the context of a subnanometer reconstruction of the 26S to arrive at a description of the RP subunit organization [23
]. Martin and colleagues took advantage of a heterologous expression system of the “lid” subcomplex [22
], locating components using maltose-binding protein (MBP) fusions and negative stain EM analyses. Combined with antibody and GST-fusion labeling of the RP “base” subcomplex, these studies directly describe the complete architecture of the proteasome RP (). This approach could in principle be applied to any new macromolecular complex lacking an extensive history of proteomic studies. More recently, and following determination of additional crystal structures of RP components [28
] daFonseca et al. [30
] have proposed a slightly modified organization for the lid subunits within a cryo-EM reconstruction of the human 26S.
Figure 2 CryoEM reconstruction of the regulatory particle (RP) of the 26S proteasome (EMDB ID: 1992) . The biochemical marker used to localize each subunit in the study by Martin and colleagues is noted, and atomic coordinates for known or homologous components (more ...)
Although crystal structures provide precise atomic information, there are frequently unstructured or dynamic regions that do not crystallize and are not visualized in the structure. Such low-complexity regions are frequent sites of post-translational modification and are commonly involved in regulating protein-protein interactions. Often times these unstructured regions become at least partially ordered in the context of a larger assembly. High-resolution cryoEM may allow visualization of these extended segments and insight into their role at interfaces. Extended segments of viral coat proteins are often involved in viral assembly and thus can be described by visualization of fully assembled viruses. One beautiful example is that described by Harrison and coworkers in the study of rotavirus VP7 protein [31
]. The authors more recently also visualized the rotavirus penetration protein VP4 in infectious particles [32
], revealing an unexpected architecture that resolved many of the perplexing questions regarding rotavirus penetration. Another example, albeit at lower resolution, is the study of microtubules interacting with the kinetochore complex Ndc80. The disordered N-terminal tail of Ndc80 mediates interactions with other Ndc80 molecules, resulting in a self-organization of the complex into clusters along microtubules. Docking of crystal structures revealed a prominent extra density not accounted for by the atomic coordinates, which extended from the N-terminus in a staggered fashion between the globular domains of the complex [33
]. Importantly, removal or phosphorylation of this segment abrogates clustering, confirming its involvement in the self-association of Ndc80 complexes.
Subnanometer resolutions like those in the examples mentioned above are not always necessary for accurate positioning of atomic structures into cryoEM density, provided there is sufficient data from other biophysical and biochemical studies. Recent work by Melero et al. reveals the pseudo-atomic architecture of the UPF surveillance complex, a central component of the nonsense-mediated decay pathway, by integrating the results from mass spectrometry, protein and nucleic acid labeling, and biochemical interaction data, into a 16Å-resolution cryoEM reconstruction [34
]. The resulting model provides a structural description of how this enzyme is stabilized at an exon junction complex, such that its helicase region of the complex is appropriately situated to remodel the 3′ end of an mRNP.
Localization of specific subunits in complexes purified from endogenous sources is commonly pursued using antibody labeling, but this approach depends on the affinity of the antibody for the epitope in the context of the assembled complex, and often suffers from substoichiometric labeling. When a recombinant expression system exists for the complex, genetic tags are a significant advantage, as demonstrated in the proteasome lid study mentioned previously. In addition to localizing a subunit by tagging one or both of its termini, internal tags can allow the effective “tracing” of the polypeptide path of large subunits. A recent implementation of this idea has been successfully utilized to effectively establish the architecture of the functional domains in human Dicer [35
]. By inserting the 15–amino acid AviTag sequence, a substrate for biotin-protein ligase, into surface loops along the structure of this enzyme, followed by biotinylation and tagging with a monovalent form of streptavidin, the protein was visualized by negative stain EM to localize the position of the extra streptavidin density.
An alternative internal tagging method recently implemented for EM labeling purposes takes advantage of the fact that the N- and C-termini of green fluorescent protein (GFP) are in close spatial proximity to one another, such that internal GFP tags, connected by a short loop, can be integrated at desired sites along a main protein chain. This strategy has been used, in combination with isotopic chemical cross-linking and mass spectrometry, to localize all subunit domains within the gene silencing complex PRC2 and generate a detailed map of interactions across the assembly (Claudio Ciferri, G.C.L. and E.N., unpublished results).