Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

doi:10.1038/ncomms9843

Cited by 256 publications

(334 citation statements)

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“…Both effects are the most severe in the capsid vertex region, limiting attainable resolution of the penton and the CATC. To further improve resolution of the vertex region, we applied a subparticle refinement and reconstruction procedure considering both local variations (74) and defocus gradient. Specifically, icosahedral orientation and center parameters of each particle image determined above were used to guide extraction of all vertex regions as subparticles with their defocus values adjusted according to their locations on each particle.…”

Section: Methodsmentioning

confidence: 99%

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

Dai

2018

Science

158

210

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INTRODUCTION Since Hippocrates first described the cutaneous spreading of herpes simplex lesions, many other diseases—chickenpox, infectious mononucleosis, nasopharyngeal carcinoma, and Kaposi’s sarcoma—have been found to be associated with the nine known human herpesviruses. Among them, herpes simplex virus type 1 (HSV-1, causes cold sores), type 2 (HSV-2, causes genital herpes), and varicella-zoster virus (causes chickenpox and shingles)—which all belong to the α-herpesvirus subfamily—can establish lifelong latent infection within our peripheral nervous system. RATIONALE A prominent feature of these neurotropic viruses is the long-range (up to tens of centimeters) axonal retrograde transport of the DNA-containing viral capsid from nerve endings at sites of infection (such as the lips) to neuronal cell bodies at the ganglia to establish latency or, upon reactivation, anterograde transport of the progeny viral particles from the ganglia to nerve terminals, resulting in reinfection of the dermis. Capsid-associated tegument complexes (CATCs) have been demonstrated to be involved in this cytoskeleton-dependent capsid transport. Because of the large size (~1300 Å) of HSV-1 particles, it has been difficult to obtain atomic structures of the HSV-1 capsid and CATC; consequently, the structural bases underlying α-herpesviruses’ remarkable capability of long-range neuronal transport and many other aspects of its life cycle are poorly understood. RESULTS By using cryo–electron microscopy, we obtained an atomic model of the HSV-1 capsid with CATC, comprising multiple conformers of the capsid proteins VP5, VP19c, VP23, and VP26 and tegument proteins pUL17, pUL25, and pUL36. Crowning every capsid vertex are five copies of heteropentameric CATC. The pUL17 monomer in each CATC bridges over triplexes Ta and Tc on the capsid surface and supports a coiled-coil helix bundle of a pUL25 dimer and a pUL36 dimer, thus positioning their flexible domains for potential involvement in nuclear egress and axonal transport of the capsid. The single C-terminal helix of pUL36 resolved in the CATC links the capsid to the outer tegument and envelope: As the largest tegument protein in all herpesviruses and essential for virion formation, pUL36 has been shown to interact extensively with other tegument proteins, which in turn interact with envelope glycoproteins. Architectural similarities between herpesvirus triplex proteins and auxiliary cementing protein gpD in bacteriophage λ, in addition to the bacteriophage HK97 gp5–like folds in their major capsid proteins and structural similarities in their DNA packaging and delivery apparatuses, indicate that the commonality between bacteriophages and herpes-viruses extends to their auxiliary components. Notwithstanding this broad evolutionary conservation, comparison of HSV-1 capsid proteins with those of other herpesviruses revealed extraordinary structural diversities in the forms of domain insertion and conformation polymorphism, not only for tegument interactions but also for DNA encapsulation...

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Section: Methodsmentioning

confidence: 99%

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

Dai

2018

Science

158

210

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“…As a control, we took advantage of a simulated dataset from a GroEL model (PDB ID code 1SS8) that was generated with crystallographic symmetry displaying a single conformation in all of the subunits (10,15,25). That dataset consisted of the same number of particles with identical Euler angles and similar signal-to-noise ratio levels compared with our experimental data but was expected to have no heterogeneity at the apical domain, being projected from a single rigid model.…”

Section: Methodsmentioning

confidence: 99%

“…Most importantly, the subunit variation might differ from particle to particle, where an asymmetric reconstruction is unlikely to yield an informative solution. Thus, we used a localized 3D classification approach (10,25) to analyze each of the particle images and determine whether computationally isolated subunits could be resolved into distinct conformations.…”

Section: Structural Variation Of the Apical Domain Within And Among Gmentioning

confidence: 99%

“…We extended the focused classification and localized reconstruction protocol (10,25) to the analysis of the subunit conformations, as described in Materials and Methods. This method allowed us to computationally isolate individual subunits per particle image and divide them into 10 classes according to their conformational similarity (Fig.…”

Section: Structural Variation Of the Apical Domain Within And Among Gmentioning

confidence: 99%

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Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

Roh

Hryc

Jeong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.nderstanding the function of a molecular machine typically requires determination of the structural components at atomic or near-atomic resolution. Although X-ray crystallography can provide atomic details, the crystal lattice forces may unnaturally constrain the structure. Moreover, solution methods, such as NMR spectroscopy, can reveal short-range dynamic behavior in the absence of information on the entire complex (1, 2). Electron cryo-microscopy (cryo-EM) has made substantial progress toward achieving atomic resolution for a broad range of molecular machines by averaging thousands to millions of single-particle images that are assumed to have identical conformations (3-6).Cryo-EM has also been used to determine multiple structures of a single specimen preparation containing conformational and compositional heterogeneity (7,8). The focused classification and refinement approaches (7, 8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9, 10) and improving feature resolvability of flexible domains through local refinement (11, 12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution. GroEL, Escherichia coli chaperonin (13,14), are composed of 14 chemically identical protomers (13,14) and have been structurally characterized by both X-ray crystallography (15, 16) and cryo-EM (5, 17-19). However, these studies have not revealed any heterogeneity among the protomers in each oligomer. Here we used a 3D classification strategy for cryo-EM data (10) to investigate structural heterogeneity among chemically identical subunits and their conformational v...

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“…When there are three or more components with mismatched symmetries, such as the corestack in the T7 phage capsid I with 5, 12, 8, and 4-fold symmetries in tandem at one of the icosahedral vertices, the combinatory assembly of these components can create a large number, for example, 5*12*8*4=1920, of states that are imaged as a mixture [55]. A focused refinement strategy is needed to zoom in on individual components to resolve them separately [55,56]. The nucleocapsid of Flaviviruses, such as Zika and Dengue, remains elusive to structural studies [21**,22*,54,57].…”

Section: Structural Complexity and Heterogeneitymentioning

confidence: 99%

Atomic cryo-EM structures of viruses

Jiang

Tang

2017

Current Opinion in Structural Biology

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During the development of single particle cryo-EM in past five decades, icosahedral viruses have led the resolution progress owing to their large mass and high symmetry. Many technical advances in cryo-EM were first established with viruses. Since reaching ~4 Å resolution in 2008, it has become a relatively routine task to solve the atomic structure of isolated viruses. The future of structural virology will be increasingly focused on remaining challenges including solving structures of jumbo viruses, intermediate functional states during assembly, maturation, and infection, and in situ structures. Recent demonstrations of near-atomic resolution structure with electron tomography and sub-tomogram averaging opens a new direction for high resolution studies of pleomorphic viruses and the pleomorphic states of icosahedral viruses that have defied past efforts using the single particle cryo-EM approach.

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Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

Cited by 256 publications

References 43 publications

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

Structure of the herpes simplex virus 1 capsid with associated tegument protein complexes

Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

Atomic cryo-EM structures of viruses

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