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...

Herpesviruses possess a genome-pressurized capsid. The 235-kilobase genome of human cytomegalovirus (HCMV) is by far the largest of any herpesvirus, yet it has been unclear how its capsid, which is similar in size to those of other herpesviruses, is stabilized. Here we report a HCMV atomic structure consisting of the herpesvirus-conserved capsid proteins MCP, Tri1, Tri2, and SCP and the HCMV-specific tegument protein pp150—totaling ~4000 molecules and 62 different conformers. MCPs manifest as a complex of insertions around a bacteriophage HK97 gp5–like domain, which gives rise to three classes of capsid floor–defining interactions; triplexes, composed of two “embracing” Tri2 conformers and a “third-wheeling” Tri1, fasten the capsid floor. HCMV-specific strategies include using hexon channels to accommodate the genome and pp150 helix bundles to secure the capsid via cysteine tetrad–to-SCP interactions. Our structure should inform rational design of countermeasures against HCMV, other herpesviruses, and even HIV/AIDS.

As key functional units in neural circuits, different types of neuronal synapses play distinct roles in brain information processing, learning, and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Here, by combining cryo-electron tomography and cryo-correlative light and electron microscopy, we distinguished intact excitatory and inhibitory synapses of cultured hippocampal neurons, and visualized the in situ 3D organization of synaptic organelles and macromolecules in their native state. Quantitative analyses of >100 synaptic tomograms reveal that excitatory synapses contain a mesh-like postsynaptic density (PSD) with thickness ranging from 20 to 50 nm. In contrast, the PSD in inhibitory synapses assumes a thin sheet-like structure ∼12 nm from the postsynaptic membrane. On the presynaptic side, spherical synaptic vesicles (SVs) of 25–60 nm diameter and discus-shaped ellipsoidal SVs of various sizes coexist in both synaptic types, with more ellipsoidal ones in inhibitory synapses. High-resolution tomograms obtained using a Volta phase plate and electron filtering and counting reveal glutamate receptor-like and GABAA receptor-like structures that interact with putative scaffolding and adhesion molecules, reflecting details of receptor anchoring and PSD organization. These results provide an updated view of the ultrastructure of excitatory and inhibitory synapses, and demonstrate the potential of our approach to gain insight into the organizational principles of cellular architecture underlying distinct synaptic functions.SIGNIFICANCE STATEMENT To understand functional properties of neuronal synapses, it is desirable to analyze their structure at molecular resolution. We have developed an integrative approach combining cryo-electron tomography and correlative fluorescence microscopy to visualize 3D ultrastructural features of intact excitatory and inhibitory synapses in their native state. Our approach shows that inhibitory synapses contain uniform thin sheet-like postsynaptic densities (PSDs), while excitatory synapses contain previously known mesh-like PSDs. We discovered “discus-shaped” ellipsoidal synaptic vesicles, and their distributions along with regular spherical vesicles in synaptic types are characterized. High-resolution tomograms further allowed identification of putative neurotransmitter receptors and their heterogeneous interaction with synaptic scaffolding proteins. The specificity and resolution of our approach enables precise in situ analysis of ultrastructural organization underlying distinct synaptic functions.

INTRODUCTION DNA replication has been studied since the 1950s. It is well established that double helical DNA needs to be separated for replication by a helicase. Each strand is then copied by a DNA polymerase, continuously on the leading and discontinuously (via Okazaki fragments) on the lagging strand, where each DNA synthesis initiates from an RNA primer provided by primase. After six decades, how DNA polymerases, helicase, primase, and their accessory factors form a replisome and perform concerted leading and lagging strand synthesis at a replication fork had never been visualized in atomic detail. RATIONALE Bacteriophage T7 presents the simplest known DNA replication system, consisting of only three proteins. Helicase and primase reside in one polypeptide chain that forms a hexamer in the presence of DNA and ATP or dTTP. T7 DNA polymerase, aided by E. coli thioredoxin as its processivity factor, carries out both leading and lagging strand DNA synthesis. Based on published biochemical data, we designed a minimal DNA fork to trap these essential proteins in replication competent states. RESULTS We determined cryogenic-electron microscopy (cryo-EM) structures of the T7 replisome and showed how its essential enzymatic functions are coordinated in three dimensions. The hexameric helicase adopts a spiral “lock washer” form that encircles the coil-like lagging DNA strand, with two nucleotides (nt) bound to each protein subunit and adjacent helicase subunits linked by domain swapping. ATP hydrolysis propels each helicase domain to translocate sequentially and coaxially along DNA in a hand-over-hand fashion, advancing 2 nt per step in the 5′ to 3′ direction (Fig. A). Instead of all enzymes moving in the same direction parallel to the downstream parental DNA, a β-hairpin from the leading-strand polymerase separates the two parental DNA strands into a T-shaped fork that enables the closely coupled helicase to unspool the downstream DNA tangentially (Fig. B). By protein-protein and DNA-mediated interactions, the leading-strand DNA polymerase and helicase cooperate to determine the rate of replication. For every ATP hydrolyzed and 2 nt advanced on DNA by the helicase, the DNA polymerase incorporates two deoxyribonucelotides. T7 primase, separated from the leading-strand polymerase by the helicase domain, synthesizes the RNA primers needed to initiate lagging-strand DNA synthesis. Transfer of a short RNA primer from the primase to DNA polymerase is facilitated by a zinc-binding-domain at the N-terminus of the T7 primase-helicase protein. Two lagging strand polymerases can be attached to the hexameric primases with one actively synthesizing DNA and the other waiting for a primer (Fig. B). Such a relay system may allow the discontinuous lagging-strand synthesis to keep pace with the leading-strand synthesis. CONCLUSION We note the similarity between hexameric DNA helicases and AAA+ protein chaperones and unfoldases, which form spiral-shaped hexamers around protein substrates, bind two amino-acid residues with each su...

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require non-native constructs involving mutations or truncations, and are challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID , a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum , an organism that has resisted conventional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and ILS complexes revealed mechanisms of 5′ splice site (ss) recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release. Here, we report a cryoEM structure of the post-catalytic P complex at 3.3Å resolution, revealing that 3′ ss is mainly recognized through non-Watson-Crick basepairing with the 5′ ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3′ exon and potentially regulating Prp22 activity. Prp22 binds nucleotides 15–21 in the 3′ exon, enabling it to pull the intron-exon or ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading or exon release, respectively.

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal “hand-shaped” polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two “helicase” domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.