DNA-Packing Portal and Capsid-Associated Tegument Complexes in the Tumor Herpesvirus KSHV.

Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an ∼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.

Cryo-EM structures of herpes simplex virus type 1 portal vertex and packaged genome.

Herpesviruses are enveloped viruses that are prevalent in the human population and are responsible for diverse pathologies, including cold sores, birth defects and cancers. They are characterized by a highly pressurized pseudo-icosahedral capsid-with triangulation number (T) equal to 16-encapsidating a tightly packed double-stranded DNA (dsDNA) genome1-3. A key process in the herpesvirus life cycle involves the recruitment of an ATP-driven terminase to a unique portal vertex to recognize, package and cleave concatemeric dsDNA, ultimately giving rise to a pressurized, genome-containing virion4,5. Although this process has been studied in dsDNA phages6-9-with which herpesviruses bear some similarities-a lack of high-resolution in situ structures of genome-packaging machinery has prevented the elucidation of how these multi-step reactions, which require close coordination among multiple actors, occur in an integrated environment. To better define the structural basis of genome packaging and organization in herpes simplex virus type 1 (HSV-1), we developed sequential localized classification and symmetry relaxation methods to process cryo-electron microscopy (cryo-EM) images of HSV-1 virions, which enabled us to decouple and reconstruct hetero-symmetric and asymmetric elements within the pseudo-icosahedral capsid. Here we present in situ structures of the unique portal vertex, genomic termini and ordered dsDNA coils in the capsid spooled around a disordered dsDNA core. We identify tentacle-like helices and a globular complex capping the portal vertex that is not observed in phages, indicative of herpesvirus-specific adaptations in the DNA-packaging process. Finally, our atomic models of portal vertex elements reveal how the fivefold-related capsid accommodates symmetry mismatch imparted by the dodecameric portal-a longstanding mystery in icosahedral viruses-and inform possible DNA-sequence recognition and headful-sensing pathways involved in genome packaging. This work showcases how to resolve symmetry-mismatched elements in a large eukaryotic virus and provides insights into the mechanisms of herpesvirus genome packaging.

Individualized predictions of early isolated distal deep vein thrombosis in patients with acute ischemic stroke: a retrospective study.

BACKGROUND: Although isolated distal deep vein thrombosis (IDDVT) is a clinical complication for acute ischemic stroke (AIS) patients, very few clinicians value it and few methods can predict early IDDVT. This study aimed to establish and validate an individualized predictive nomogram for the risk of early IDDVT in AIS patients. METHODS: This study enrolled 647 consecutive AIS patients who were randomly divided into a training cohort (n = 431) and a validation cohort (n = 216). Based on logistic analyses in training cohort, a nomogram was constructed to predict early IDDVT. The nomogram was then validated using area under the receiver operating characteristic curve (AUROC) and calibration plots. RESULTS: The multivariate logistic regression analysis revealed that age, gender, lower limb paralysis, current pneumonia, atrial fibrillation and malignant tumor were independent risk factors of early IDDVT; these variables were integrated to construct the nomogram. Calibration plots revealed acceptable agreement between the predicted and actual IDDVT probabilities in both the training and validation cohorts. The nomogram had AUROC values of 0.767 (95% CI: 0.742-0.806) and 0.820 (95% CI: 0.762-0.869) in the training and validation cohorts, respectively. Additionally, in the validation cohort, the AUROC of the nomogram was higher than those of the other scores for predicting IDDVT. CONCLUSIONS: The present nomogram provides clinicians with a novel and easy-to-use tool for the prediction of the individualized risk of IDDVT in the early stages of AIS, which would be helpful to initiate imaging examination and interventions timely.

Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy.

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.

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument.

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the α-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the γ-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.

Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa.

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8-14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF from Microcystis aeruginosa PCC 7806 is reported at 2.7 Šresolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+ß class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

Overcoming the preferred orientation problem in cryoEM with self-supervised deep-learning.

While advances in single-particle cryoEM have enabled the structural determination of macromolecular complexes at atomic resolution, particle orientation bias (the so-called "preferred" orientation problem) remains a complication for most specimens. Existing solutions have relied on biochemical and physical strategies applied to the specimen and are often complex and challenging. Here, we develop spIsoNet, an end-to-end self-supervised deep-learning-based software to address the preferred orientation problem. Using preferred-orientation views to recover molecular information in under-sampled views, spIsoNet improves both angular isotropy and particle alignment accuracy during 3D reconstruction. We demonstrate spIsoNet's capability of generating near-isotropic reconstructions from representative biological systems with limited views, including ribosomes, ß-galactosidases, and a previously intractable hemagglutinin trimer dataset. spIsoNet can also be generalized to improve map isotropy and particle alignment of preferentially oriented molecules in subtomogram averaging. Therefore, without additional specimen-preparation procedures, spIsoNet provides a general computational solution to the preferred orientation problem.

The incredible bulk: Human cytomegalovirus tegument architectures uncovered by AI-empowered cryo-EM.

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses' capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. We use deep-learning-enhanced cryogenic electron microscopy to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show that pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head-linker-capsid-binding motif conducive to PVAT reconfiguration, and pUL47/48 dimers form 45-nm-long filaments extending from the portal vertex. These results provide a structural framework for understanding how herpesvirus tegument facilitates and evolves during processes spanning viral genome packaging to delivery.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy - rationalizing the use of surfactant, which is a direct solution to reducing the surface tension of the aqueous solution. By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that applicable surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple, and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, the ClC-1 channel, has led to its first near-atomic structure using cryoEM.

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM.

As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids. However, the reasons for such misbehavior are not well understood, which limits systematic approaches to solving the problem. Here, we have developed a theoretical formulation that explains these observations. Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy-rationalizing the use of surfactant, which is a direct solution to reduce the surface tension of the aqueous solution. By performing cryogenic electron tomography (cryoET) on the widely-tested sample, GroEL, we demonstrate that, in a standard buffer solution, nearly all particles migrate to the AWI. Gradually reducing the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that suitable surfactants do not damage the biological complex, thus suggesting that they might provide a practical, simple, and general solution to the problem for high-resolution cryoEM. Applying this solution to a real-world AWI adsorption problem involving a more challenging membrane protein, namely, the ClC-1 channel, has resulted in its near-atomic structure determination using cryoEM.

[Dissolved Ion Concentrations and Isotope Values in Agricultural Fertilizer Locally Applied in Henan Province].

Agricultural fertilizers (AFs) have provided vegetation with necessary nutrients, but unabsorbed constituents have been retarded in soil, potentially affecting the quality of adjacent surface water and groundwater. AFs element contents and stable isotope compositions have often been utilized to assess and calculate AFs pollution to nitrate and sulfate in surface water and groundwater; however, due to various AFs applied, the dissolved ion concentrations and isotope ratios are still unknown. This study collected commercial AF widely utilized in Henan province, China, to constrain their ion concentrations and isotope values. The dissolved ions (1 g AFs dissolved in 1 L ultrapure water), sulfate sulfur, and oxygen isotope values(δ34S and δ18O) were analyzed, and total nitrogen (TN) contents coupled with nitrogen isotope values(δ15N) in solid AFs were determined to elucidate their elemental and isotopic compositions. These characteristics provided a scientific basis for further assessing their contributions to surface water and groundwater contaminations. The results indicated that pH values in the AFs solutions varied from 3.6 to 10.2, with a mean value of 6.7±1.5 (n=30, 1σ). Sulfate (SO42-) and nitrate (NO3-) concentrations ranged from 4.38 mg·L-1 to 827.29 mg·L-1 and from 1.34 mg·L-1 to 208.90 mg·L-1, with median values of 192.80 mg·L-1 and 13.51 mg·L-1 and average values of (256.19±239.83) mg·L-1 (n=30) and (37.07±53.21) mg·L-1 (n=29), respectively. Dissolved sulfate δ34S and δ18O values in AFs varied from -3.5‰ to 19.0‰ and from 6.7‰ to 18.5‰, with median values of 4.1‰ and 10.1‰ and mean values of (5.8±5.5)‰ (n=22, 1σ) and (10.7±2.7)‰ (n=22, 1σ), respectively. TN and δ15N values in AFs ranged from 0.5% to 38.9% and from -2.7‰ to 3.4‰, with median values of 13.3% and 0.0‰ and average values of (14.8±9.3)% (n=25) and 0.0±1.5‰ (n=24, 1σ), respectively. The lower averaged δ34S values and positive averaged δ18O values potentially resulted from sulfuric acids added as raw materials, giving rise to a negative relationship between pH values and SO42- concentrations (P<0.05). The δ15N values of AFs were close to that of air N2, corresponding to the fact that NO3--N and NH4+-N were synthesized via air N2. Our results revealed the dissolved ion concentrations of SO42-, NO3-, and NH4+ and their δ34S, δ18O, and δ15N values of typically applied AFs in Henan province, which provided the scientific basis for studying the AFs contributions to SO42- and NO3- pollutions in surface water and groundwater surroundings.

Discovery, structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase.

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent mitochondrial enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments, and determined the structures of different filament regions at 3.4, 3.9, and 7.3 Å resolution using cryoEM. α6ß6 LtMCCs assemble in a twisted-stacks architecture, manifesting as supramolecular rods up to 400 nm. Filamentous LtMCCs bind biotin non-covalently and lack coenzyme A. Filaments elongate by stacking α6ß6 LtMCCs onto the exterior α-trimer of the terminal LtMCC. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the function of polymerization in the carboxylase superfamily and beyond.

CryoEM reveals oligomeric isomers of a multienzyme complex and assembly mechanics.

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six α-subunits and six ß-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC ß-subunits form stable homohexamers, to which different numbers of α-subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulae α0ß6, α1ß6, α2ß6, α3ß6, α4ß6, α5ß6, α6ß6) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

Preparation and characterization of carboxymethyl chitosan/pullulan composite film incorporated with eugenol and its application in the preservation of chilled meat.

To solve the problem of easy spoilage of chilled meat during storage, we fabricated a novel composite film using carboxymethyl chitosan (CMCS)/pullulan (Pul)/eugenol (E) by casting method. The results showed that the mechanical properties of the films were better when the CMCS/Pul ratio was 2.5/2.5. The Fourier transform infrared spectroscopy (FTIR) results showed that intermolecular hydrogen bonds were formed among E, CMCS, and Pul, which was consistent with the rheological test results. Scanning electron microscopic (SEM) images showed that eugenol was well dispersed in the CMCS/Pul matrix. The addition of eugenol significantly increased the antibacterial properties and antioxidant properties. Moreover, when 5% eugenol was added, the water vapor permeability (WVP) of the film reduced to 2.41 × 10-11 g/m·s·Pa. Finally, the freshness of the chilled meat wrapped with the eugenol-containing composite film was prolonged, thereby offering a potential alternative to synthetic materials.

Digitalizing neuronal synapses with cryo-electron tomography and correlative microscopy.

Synapses are the structural and functional joints of neuronal circuits, and brain function is fundamentally based on synaptic quantal transmission and plasticity. Precise mapping of key components within individual synapses in different states can reveal the principles governing synapse formation, transmission, and plasticity and improving understanding of the mechanisms of synapse-related diseases. Cryo-electron tomography (cryo-ET) and correlative microscopy are increasingly powerful tools that can dissect the molecular sociology of intact cells, including neuronal synapses. In this study, we discuss current progress made in cryo-ET studies assessing neuronal synapses, especially sample preparation, molecule identification, and correlative approaches for synaptic dynamics and functions.

Isotropic reconstruction for electron tomography with deep learning.

Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic "missing-wedge" problem has presented major challenges in visualization and interpretation of tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs the missing-wedge information and increases signal-to-noise ratio, using the knowledge learned from raw tomograms. Without the need for sub-tomogram averaging, IsoNet generates tomograms with significantly reduced resolution anisotropy. Applications of IsoNet to three representative types of cryoET data demonstrate greatly improved structural interpretability: resolving lattice defects in immature HIV particles, establishing architecture of the paraflagellar rod in Eukaryotic flagella, and identifying heptagon-containing clathrin cages inside a neuronal synapse of cultured cells. Therefore, by overcoming two fundamental limitations of cryoET, IsoNet enables functional interpretation of cellular tomograms without sub-tomogram averaging. Its application to high-resolution cellular tomograms should also help identify differently oriented complexes of the same kind for sub-tomogram averaging.

Study on Bulk Texture and Mechanical Properties of As-Extruded Wide Mg-Al-Zn Alloy Sheets with Different Al Addition.

The wide Mg alloy sheets produced by hot extrusion usually can easily form an inhomogeneous texture, resulting in anisotropic mechanical properties and poor formability. However, few studies have been carried out on the bulk texture investigation at different areas of as-extruded Mg alloy sheets, especially the Mg alloys with different alloying elements. In this work, the effect of Al on the bulk texture and mechanical properties at different areas for three wide Mg-Al-Zn alloy sheets with different Al contents (Mg-3Al-0.5Zn, Mg-8Al-0.5Zn and Mg-9Al-0.5Zn) are mainly investigated by neutron diffraction. The results showed that a strong and uneven basal texture was formed in the Mg-3Al-0.5Zn sheet. Meanwhile, the intensity of the basal texture was significantly weakened due to the numerous fine precipitates of Mg17Al12 particles, with the Al content increasing, which hinder the grain growth during extrusion, while fine recrystallized grains have a more random orientation. The enhanced tensile properties in Mg-8Al-0.5Zn and Mg-9Al-0.5Zn alloy sheets are ascribed to the cooperation effect of a refined microstructure, precipitates and weakened basal texture. Among the three Mg alloy sheets, the Mg-8Al-0.5Zn alloy sheet has a yield strength of about 270 MPa, an ultimate tensile strength of about 330 MPa and ultimate elongation of about 16% in the extrusion direction, which possesses the most excellent comprehensive mechanical properties.

Structure of human cytomegalovirus virion reveals host tRNA binding to capsid-associated tegument protein pp150.

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.

Mesophasic organization of GABAA receptors in hippocampal inhibitory synapses.

Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABAARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.