ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1-vectored HexaPro-stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

Structure and activity of particulate methane monooxygenase arrays in methanotrophs.

Methane-oxidizing bacteria play a central role in greenhouse gas mitigation and have potential applications in biomanufacturing. Their primary metabolic enzyme, particulate methane monooxygenase (pMMO), is housed in copper-induced intracytoplasmic membranes (ICMs), of which the function and biogenesis are not known. We show by serial cryo-focused ion beam (cryoFIB) milling/scanning electron microscope (SEM) volume imaging and lamellae-based cellular cryo-electron tomography (cryoET) that these ICMs are derived from the inner cell membrane. The pMMO trimer, resolved by cryoET and subtomogram averaging to 4.8 Å in the ICM, forms higher-order hexagonal arrays in intact cells. Array formation correlates with increased enzymatic activity, highlighting the importance of studying the enzyme in its native environment. These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context.

Application of super-resolution and correlative double sampling in cryo-electron microscopy.

Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that collecting cryo-ET STA data using the same conditions as SPA, with both correlated double sampling (CDS) and the super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in the super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in the super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising the data size on disk and the area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

Cryo-electron Tomography Remote Data Collection and Subtomogram Averaging.

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.

GaS:WS2 Heterojunctions for Ultrathin Two-Dimensional Photodetectors with Large Linear Dynamic Range across Broad Wavelengths.

Two-dimensional (2D) photodetectors based on photovoltaic effect or photogating effect can hardly achieve both high photoresponsivity and large linear dynamic range at the same time, which greatly limits many practical applications such as imaging sensors. Here, the conductive-sensitizer strategy, a general design for improving photoresponsivity and linear dynamic range in 2D photodetectors is provided and experimentally demonstrated on vertically stacked bilayer WS2/GaS0.87 under a parallel circuit mode. Owing to successful band alignment engineering, the isotype type-II heterojunction enables efficient charge carrier transfer from WS2, the high-mobility sensitizer, to GaS0.87, the low-mobility channel, under illumination from a broad visible spectrum. The transferred electron charges introduce a reverse electric field which efficiently lowers the band offset between the two materials, facilitating a transition from low-mobility photocarrier transport to high-mobility photocarrier transport with increasing illumination power. We achieved a large linear dynamic range of 73 dB as well as a high and constant photoresponsivity of 13 A/W under green light. X-ray photoelectron spectroscopy, cathodoluminescence, and Kelvin probe force microscopy further identify the key role of defects in monolayer GaS0.87 in engineering the band alignment with monolayer WS2. This work proposes a design route based on band and interface modulation for improving performance of 2D photodetectors and provides deep insights into the important role of strong interlayer coupling in offering heterostructures with desired properties and functions.

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events â€" e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

Atomic structure and defect dynamics of monolayer lead iodide nanodisks with epitaxial alignment on graphene.

Lead Iodide (PbI2) is a large bandgap 2D layered material that has potential for semiconductor applications. However, atomic level study of PbI2 monolayer has been limited due to challenges in obtaining thin crystals. Here, we use liquid exfoliation to produce monolayer PbI2 nanodisks (30-40 nm in diameter and > 99% monolayer purity) and deposit them onto suspended graphene supports to enable atomic structure study of PbI2. Strong epitaxial alignment of PbI2 monolayers with the underlying graphene lattice occurs, leading to a phase shift from the 1 T to 1 H structure to increase the level of commensuration in the two lattice spacings. The fundamental point vacancy and nanopore structures in PbI2 monolayers are directly imaged, showing rapid vacancy migration and self-healing. These results provide a detailed insight into the atomic structure of monolayer PbI2, and the impact of the strong van der Waals interaction with graphene, which has importance for future applications in optoelectronics.

Controlling Defects in Continuous 2D GaS Films for High-Performance Wavelength-Tunable UV-Discriminating Photodetectors.

A chemical vapor deposition method is developed for thickness-controlled (one to four layers), uniform, and continuous films of both defective gallium(II) sulfide (GaS): GaS0.87 and stoichiometric GaS. The unique degradation mechanism of GaS0.87 with X-ray photoelectron spectroscopy and annular dark-field scanning transmission electron microscopy is studied, and it is found that the poor stability and weak optical signal from GaS are strongly related to photo-induced oxidation at defects. An enhanced stability of the stoichiometric GaS is demonstrated under laser and strong UV light, and by controlling defects in GaS, the photoresponse range can be changed from vis-to-UV to UV-discriminating. The stoichiometric GaS is suitable for large-scale, UV-sensitive, high-performance photodetector arrays for information encoding under large vis-light noise, with short response time (<66 ms), excellent UV photoresponsivity (4.7 A W-1 for trilayer GaS), and 26-times increase of signal-to-noise ratio compared with small-bandgap 2D semiconductors. By comprehensive characterizations from atomic-scale structures to large-scale device performances in 2D semiconductors, the study provides insights into the role of defects, the importance of neglected material-quality control, and how to enhance device performance, and both layer-controlled defective GaS0.87 and stoichiometric GaS prove to be promising platforms for study of novel phenomena and new applications.

Controlling Photoluminescence Enhancement and Energy Transfer in WS2 :hBN:WS2 Vertical Stacks by Precise Interlayer Distances.

2D semiconducting transition metal dichalcogenides (TMDs) are endowed with fascinating optical properties especially in their monolayer limit. Insulating hBN films possessing customizable thickness can act as a separation barrier to dictate the interactions between TMDs. In this work, vertical layered heterostructures (VLHs) of WS2 :hBN:WS2 are fabricated utilizing chemical vapor deposition (CVD)-grown materials, and the optical performance is evaluated through photoluminescence (PL) spectroscopy. Apart from the prohibited indirect optical transition due to the insertion of hBN spacers, the variation in the doping level of WS2 drives energy transfer to arise from the layer with lower quantum efficiency to the other layer with higher quantum efficiency, whereby the total PL yield of the heterosystem is increased and the stack exhibits a higher PL intensity compared to the sum of those in the two WS2 constituents. Such doping effects originate from the interfaces that WS2 monolayers reside on and interact with. The electron density in the WS2 is also controlled and subsequent modulation of PL in the heterostructure is demonstrated by applying back-gated voltages. Other influential factors include the strain in WS2 and temperature. Being able to tune the energy transfer in the VLHs may expand the development of photonic applications in 2D systems.

Ultrathin All-2D Lateral Graphene/GaS/Graphene UV Photodetectors by Direct CVD Growth.

UV-sensitive lateral all-two-dimensional (2D) photodetecting devices are produced by growing the large band gap layered GaS between graphene electrode pairs directly using chemical vapor deposition methods. The use of prepatterned graphene electrode pairs on the Si wafer enables more than 200 devices to be fabricated simultaneously. We show that the surface chemistry of the substrate during GaS leads to selective growth in graphene gaps, forming the lateral heterostructures, rather than on the surface of graphene. The graphene/GaS/graphene lateral photodetecting devices are demonstrated to be sensitive to UV light only, with no measurable response to visible light. Furthermore, we demonstrate UV-band discrimination in photosensing, with measured photocurrents only produced for middle-UV and not for near-UV wavelength regions. The detection limit could reach down to 2.61 µW/cm2 with a photoresponsivity as high as 11.7 A/W and a photo gain of 53.7 under 270 nm excitation. Gate-dependent modulation of the photocurrent is also demonstrated. The photodetectors exhibit long-term stability and reproducible ON-OFF switching behavior, with a response time lower than 60 ms. These results provide insights into how ultrathin UV sensing devices can be created using only 2D materials by exploiting large band gap 2D semiconductors such as GaS.

Direct Laser Patterning and Phase Transformation of 2D PdSe2 Films for On-Demand Device Fabrication.

Heterophase homojunction formation in atomically thin 2D layers is of great importance for next-generation nanoelectronics and optoelectronics applications. Technologically challenging, controllable transformation between the semiconducting and metallic phases of transition metal chalcogenides is of particular importance. Here, we demonstrate that controlled laser irradiation can be used to directly ablate PdSe2 thin films using high power or trigger the local transformation of PdSe2 into a metallic phase PdSe2-x using lower laser power. Such transformations are possible due to the low decomposition temperature of PdSe2 and a variety of stable phases compared to other 2D transition metal dichalcogenides. Scanning transmission electron microscopy is used to reveal the laser-induced Se-deficient phases of PdSe2 material. The process sensitivity to the laser power allows patterning flexibility for resist-free device fabrication. The laser-patterned devices demonstrate that a laser-induced metallic phase PdSe2-x is stable with increased conductivity by a factor of about 20 compared to PdSe2. These findings contribute to the development of nanoscale devices with homojunctions and scalable methods to achieve structural transformations in 2D materials.

Photocurrent Direction Control and Increased Photovoltaic Effects in All-2D Ultrathin Vertical Heterostructures Using Asymmetric h-BN Tunneling Barriers.

Two-dimensional (2D) materials are atomically thick and without out-of-plane dangling bonds. As a result, they could break the confinement of lattice matching, and thus can be freely mixed and matched together to construct vertical van der Waals heterostructures. Here, we demonstrated an asymmetrical vertical structure of graphene/hexagonal boron nitride (h-BN)/tungsten disulfide (WS2)/graphene using all chemical vapor deposition grown 2D materials. Three building blocks are utilized in this construction: conductive graphene as a good alternative for the metal electrode due to its tunable Fermi level and ultrathin nature, semiconducting transition-metal dichalcogenides (TMDs) as an ultrathin photoactive material, and insulating h-BNas a tunneling barrier. Such an asymmetrical vertical structure exhibits a much stronger photovoltaic effect than the symmetrical vertical one without h-BN. By changing the sequence of h-BN in the vertical stack, we could even control the electron flow direction. Also, improvement has been further made by increasing the thickness of h-BN. The photovoltaic effect is attributed to different possibilities of excited electrons on TMDs to migrate to top and bottom graphene electrodes, which is caused by potential differences introduced by an insulating h-BN layer. This study shows that h-BN could be effectively used as a tunneling barrier in the asymmetrical vertical heterostructure to improve photovoltaic effect and control the electron flow direction, which is crucial for the design of other 2D vertical heterostructures to meet various needs of electronic and optoelectronic devices.

Postgrowth Substitutional Tin Doping of 2D WS2 Crystals Using Chemical Vapor Deposition.

Doping of two-dimensional materials provides them tunable physical properties and widens their applications. Here, we demonstrate the postgrowth doping strategy in monolayer and bilayer tungsten disulfide (WS2) crystals, which utilizes a metal exchange mechanism, whereby Sn atoms become substitutional dopants in the W sites by energetically favorable replacement. We achieve this using chemical vapor deposition techniques, where high-quality grown WS2 single crystals are first grown and then subsequently reacted with a SnS precursor. Thermal control of the exchange doping mechanism is revealed, indicating that a sufficiently high enough temperature is required to create the S vacancies that are the initial binding sites for the SnS precursor and metal exchange occurrence. This results in a better control of dopant distribution compared to the tradition all-in-one approach, where dopants are added during the growth phase. The Sn dopants exhibit an n-type doping behavior in the WS2 layers based on the decreased threshold voltage obtained from transistor device measurements. Annular dark-field scanning transmission electron microscopy shows that in bilayer WS2 the Sn doping occurs only in the top layer, creating vertical heterostructures with atomic layer doping precision. This postgrowth modification opens up ways to selectively dope one layer at a time and construct mixed stoichiometry vertical heterojunctions in bilayer crystals.

Metal Atom Markers for Imaging Epitaxial Molecular Self-Assembly on Graphene by Scanning Transmission Electron Microscopy.

Direct imaging of single molecules has to date been primarily achieved using scanning probe microscopy, with limited success using transmission electron microscopy due to electron beam damage and low contrast from the light elements that make up the majority of molecules. Here, we show single complex molecule interactions can be imaged using annular dark field scanning TEM (ADF-STEM) by inserting heavy metal markers of Pt atoms and detecting their positions. Using the high angle ADF-STEM Z1.7 contrast, combined with graphene as an electron transparent support, we track the 2D monolayer self-assembly of solution-deposited individual linear porphyrin hexamer (Pt-L6) molecules and reveal preferential alignment along the graphene zigzag direction. The epitaxial interactions between graphene and Pt-L6 drive a reduction in the interporphyrin distance to allow perfect commensuration with the graphene. These results demonstrate how single metal atom markers in complex molecules can be used to study large scale packing and chain bending at the single molecule level.

High-Performance WS2 Monolayer Light-Emitting Tunneling Devices Using 2D Materials Grown by Chemical Vapor Deposition.

The solid progress in the study of a single two-dimensional (2D) material underpins the development for creating 2D material assemblies with various electronic and optoelectronic properties. We introduce an asymmetric structure by stacking monolayer semiconducting tungsten disulfide, metallic graphene, and insulating boron nitride to fabricate numerous red channel light-emitting devices (LEDs). All the 2D crystals were grown by chemical vapor deposition (CVD), which has great potential for future industrial scale-up. Our LEDs exhibit visibly observable electroluminescence (EL) at both 5.5 V forward and 7.0 V backward biasing, which correlates well with our asymmetric design. The red emission can last for at least several minutes, and the success rate of the working device that can emit detectable EL is up to 80%. In addition, we show that sample degradation is prone to happen when a continuing bias, much higher than the threshold voltage, is applied. Our success of using high-quality CVD-grown 2D materials for red light emitters is expected to provide the basis for flexible and transparent displays.

High Photoresponsivity in Ultrathin 2D Lateral Graphene:WS2:Graphene Photodetectors Using Direct CVD Growth.

We show that reducing the degree of van der Waals overlapping in all 2D ultrathin lateral devices composed of graphene:WS2:graphene leads to significant increase in photodetector responsivity. This is achieved by directly growing WS2 using chemical vapor deposition (CVD) in prepatterned graphene gaps to create epitaxial interfaces. Direct-CVD-grown graphene:WS2:graphene lateral photodetecting transistors exhibit high photoresponsivities reaching 121 A/W under 2.7 × 105 mW/cm2 532 nm illumination, which is around 2 orders of magnitude higher than similar devices made by the layer-by-layer transfer method. The photoresponsivity of our direct-CVD-grown device shows negative correlation with illumination power under different gate voltages, which is different from similar devices made by the transfer method. We show that the high photoresponsivity is due to the lowering of effective Schottky barrier height by improving the contact between graphene and WS2. Furthermore, the direct CVD growth reduces overlapping sections of WS2:Gr and leads to more uniform lateral systems. This approach provides insights into scalable manufacturing of high-quality 2D lateral electronic and optoelectronic devices.

MoS2 Liquid Cell Electron Microscopy Through Clean and Fast Polymer-Free MoS2 Transfer.

Two dimensional (2D) materials have found various applications because of their unique physical properties. For example, graphene has been used as the electron transparent membrane for liquid cell transmission electron microscopy (TEM) due to its high mechanical strength and flexibility, single-atom thickness, chemical inertness, etc. Here, we report using 2D MoS2 as a functional substrate as well as the membrane window for liquid cell TEM, which is enabled by our facile and polymer-free MoS2 transfer process. This provides the opportunity to investigate the growth of Pt nanocrystals on MoS2 substrates, which elucidates the formation mechanisms of such heterostructured 2D materials. We find that Pt nanocrystals formed in MoS2 liquid cells have a strong tendency to align their crystal lattice with that of MoS2, suggesting a van der Waals epitaxial relationship. Importantly, we can study its impact on the kinetics of the nanocrystal formation. The development of MoS2 liquid cells will allow further study of various liquid phenomena on MoS2, and the polymer-free MoS2 transfer process will be implemented in a wide range of applications.