A cryo-TSEM with temperature cycling capability allows deep sublimation of ice to uncover fine structures in thick cells.

The scanning electron microscope (SEM) has been reassembled into a new type of cryo-electron microscope (cryo-TSEM) by installing a new cryo-transfer holder and anti-contamination trap, which allowed simultaneous acquisition of both transmission images (STEM images) and surface images (SEM images) in the frozen state. The ultimate temperatures of the holder and the trap reached - 190 °C and - 210 °C, respectively, by applying a liquid nitrogen slush. The STEM images at 30 kV were comparable to, or superior to, the images acquired with conventional transmission electron microscope (100 kV TEM) in contrast and sharpness. The unroofing method was used to observe membrane cytoskeletons instead of the frozen section and the FIB methods. Deep sublimation of ice surrounding unroofed cells by regulating temperature enabled to emerge intracellular fine structures in thick frozen cells. Hence, fine structures in the vicinity of the cell membrane such as the cytoskeleton, polyribosome chains and endoplasmic reticulum (ER) became visible. The ER was distributed as a wide, flat structure beneath the cell membrane, forming a large spatial network with tubular ER.

Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2.

Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.

Inhibitory Effect of Osthole from Cnidium monnieri on Tobacco Mosaic Virus (TMV) Infection in Nicotiana glutinosa.

The coumarin compound of osthole was extracted from Cnidium monnieri and identified by LC-MS and 1H- and 13C-NMR. Osthole was tested for anti-virus activity against tobacco mosaic virus (TMV) using the half-leaf method. The results showed that stronger antiviral activity on TMV infection appeared in Nicotiana glutinosa than that of eugenol and ningnanmycin, with inhibitory, protective, and curative effects of 72.57%, 70.26%, and 61.97%, respectively. Through observation of the TMV particles, we found that osthole could directly affect the viral particles. Correspondingly, the level of coat protein detected by Western blot was significantly reduced when the concentrations of osthole increased in tested plants compared to that of the control. These results suggest that osthole has anti-TMV activity and may be used as a biological reagent to control the plant virus in the half-leaf method.

A single-molecule atomic force microscopy study reveals the antiviral mechanism of tannin and its derivatives.

Antiviral agents work by stopping or intervening the virus replication. Virus replication is a fast and multi-step process while effective antiviral intervention requires agents to interact with the protein coat, genetic RNA/DNA or both during virus replication. Thus, quantifying these interactions at the molecular level, although it is quite challenging, is very important for an understanding of the underlying molecular mechanism of antiviral intervention. Here, at the single molecule level, we employ single molecule force spectroscopy (SMFS) in combination with AFM imaging and choose tobacco mosaic virus (TMV)/tannin as a model system of tubular virus to directly study how the inhibitor influences the interactions of RNA and coat protein. We illustrated the antiviral mechanism of tannin during the three main stages of TMV infection, i.e., before the entry of cells, the disassembly of genetic RNA and reassembly of genetic RNA, respectively. Our SMFS results show that tannin and its derivatives can stabilize the TMV complex by enhancing the interactions between RNA and coat protein via weak interactions, such as hydrogen bonding and hydrophobic interactions. In addition, the stabilization effect showed molecular weight dependence, i.e., for higher molecular weight tannin the stabilization occurs after genetic RNA gets partially disassembled from the protein coat, while the lower molecular weight tannin hydrolyte starts experiencing the stabilization effect before the RNA disassembly. Furthermore, the cycling stretching-relaxation experiments in the presence/absence of tannin proved that tannin can prevent the assembling of RNA and coat protein. In addition, the AFM imaging results demonstrate that tannin can cause the aggregation of TMV particles in a concentration-dependent manner; a higher concentration of tannin will cause more severe aggregations. These results deepen our understanding of the antiviral mechanism of tannin and its derivatives, which facilitate the rational design of efficient agents for antiviral therapy.

CM01: a facility for cryo-electron microscopy at the European Synchrotron.

Recent improvements in direct electron detectors, microscope technology and software provided the stimulus for a `quantum leap' in the application of cryo-electron microscopy in structural biology, and many national and international centres have since been created in order to exploit this. Here, a new facility for cryo-electron microscopy focused on single-particle reconstruction of biological macromolecules that has been commissioned at the European Synchrotron Radiation Facility (ESRF) is presented. The facility is operated by a consortium of institutes co-located on the European Photon and Neutron Campus and is managed in a similar fashion to a synchrotron X-ray beamline. It has been open to the ESRF structural biology user community since November 2017 and will remain open during the 2019 ESRF-EBS shutdown.

TMV Particles: The Journey From Fundamental Studies to Bionanotechnology Applications.

Ever since its initial characterization in the 19th century, tobacco mosaic virus (TMV) has played a prominent role in the development of modern virology and molecular biology. In particular, research on the three-dimensional structure of the virus particles and the mechanism by which these assemble from their constituent protein and RNA components has made TMV a paradigm for our current view of the morphogenesis of self-assembling structures, including viral particles. More recently, this knowledge has been applied to the development of novel reagents and structures for applications in biomedicine and bionanotechnology. In this article, we review how fundamental science has led to TMV being at the vanguard of these new technologies.

Synthesis of Metal-Organic Frameworks on Tobacco Mosaic Virus Templates.

Tobacco mosaic virus (TMV) has long been exploited as a robust biological scaffold for organic/inorganic modification owing to its anisotropic structure and chemically addressable amino acid residues on both the exterior and interior. We present the fabrication of a crystalline microporous metal-organic framework (MOF) shell on the exterior of TMV, which retains its rod-like morphology, and produces uniformly formed core-shell structures with high accessible surface area and pore volume. We also describe an exfoliation method that can recover the intact viral particle from the core-shell composite.

TMV Disk Scaffolds for Making sub-30 nm Silver Nanorings.

Nanosized bioscaffolds can be utilized to tackle the challenge of size reduction of metallic rings owing to their miniature features as well as their well-known biomineralization capacity. The tobacco mosaic virus coat protein is used as a command surface to grow and assemble silver nanoparticles into sub-30 nm rings. The versatility of TMV allows the formation of both solid silver rings and rings consisting of discrete silver nanoparticles. The pH-dependent coulombic surface map along with the annular geometry of the protein aggregate allow the generation of rings with or without a central nanoparticle. Our silver rings are believed to be the smallest to date, and they can offer a test material for existing theories on metallic nanorings of this heretofore unreached size scale.

Self-Assembly of Protein Crystals with Different Crystal Structures Using Tobacco Mosaic Virus Coat Protein as a Building Block.

In this work, a typical cylinder-shaped tobacco mosaic virus coat protein (TMVCP) is employed as an anisotropic building block to assemble into triclinic and hexagonal close-packed (HCP) protein crystals by introducing cysteine residues at the 1 and 3 sites and four histidine residues at the C-terminal, respectively. The engineered functional groups of cysteine and histidine in the TMVCP and the self-assembly conditions determine the thermodynamics and kinetics in the self-assembly process for forming different crystal structures. The results show that the TMVCPs are thermodynamically driven to form triclinic crystals due to the formation of disulfide bonds between neighboring TMVCPs. On the other hand, the self-assembly of HCP crystals is kinetically directed by the strong metal-histidine chelation. This work not only greatly expands TMVCP for fabricating promising nanomaterials but also represents an approach to adjusting the protein crystal structures by tuning the thermodynamics and kinetics during crystallization.

Cooperative colloidal self-assembly of metal-protein superlattice wires.

Material properties depend critically on the packing and order of constituent units throughout length scales. Beyond classically explored molecular self-assembly, structure formation in the nanoparticle and colloidal length scales have recently been actively explored for new functions. Structure of colloidal assemblies depends strongly on the assembly process, and higher structural control can be reliably achieved only if the process is deterministic. Here we show that self-assembly of cationic spherical metal nanoparticles and anionic rod-like viruses yields well-defined binary superlattice wires. The superlattice structures are explained by a cooperative assembly pathway that proceeds in a zipper-like manner after nucleation. Curiously, the formed superstructure shows right-handed helical twisting due to the right-handed structure of the virus. This leads to structure-dependent chiral plasmonic function of the material. The work highlights the importance of well-defined colloidal units when pursuing unforeseen and complex assemblies.Colloidal self-assembly is a unique method to produce three-dimensional materials with well-defined hierarchical structures and functionalities. Liljeström et al. show controlled preparation of macroscopic chiral wires with helical plasmonic superlattice structure composed of metal nanoparticles and viruses.

Towards a correlative approach for characterising single virus particles by transmission electron microscopy and nanoscale Raman spectroscopy.

The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.

Three-dimensional CTF correction improves the resolution of electron tomograms.

Correction of the contrast transfer function (CTF) of the microscope is a necessary step, in order to achieve high resolution from averaged electron microscopic images. Thereby, the CTF is first estimated and subsequently the electron micrograph is corrected, so that the negative oscillations of the CTF are equalized. Typically, the CTF correction is performed in 2D and the tilt-induced focus gradient is taken into account. Most often, the sample-thickness-induced focus gradient is ignored. Theoretical considerations, as well as implementation suggestions, for a 3D CTF correction that considers both gradients have been proposed before, although an implementation achieving a resolution improvement has been lacking, primarily due to computational reasons. Here, we present a comprehensive solution for a 3D CTF correction based on the Jensen-Kornberg scheme, which performs a slice-by-slice correction of the CTF within the tomographic reconstruction. We show that the computational requirements are comparable to those of 2D CTF correction. Using the examples of mitochondrial ribosomes and tobacco mosaic virus we demonstrate the improvement of the reconstruction quality with the 3D CTF correction, and the resolution gain on sub-tomogram averaging. More interestingly, for tomographic applications, the quality of the individual sub-tomograms before averaging increases significantly. We find that 3D CTF correction always produces equal or better results than 2D CTF correction.

Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.

A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers.

Key factors of scanning a plant virus with AFM in air and aqueous solution.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A0 ) and the amplitude set-point (A) are also assessed here. While high A0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 N m-1 ) and high A0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A0 = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm. Microsc. Res. Tech. 80:18-29, 2017. © 2016 Wiley Periodicals, Inc.

Cryo-EM Structure Determination Using Segmented Helical Image Reconstruction.

Treating helices as single-particle-like segments followed by helical image reconstruction has become the method of choice for high-resolution structure determination of well-ordered helical viruses as well as flexible filaments. In this review, we will illustrate how the combination of latest hardware developments with optimized image processing routines have led to a series of near-atomic resolution structures of helical assemblies. Originally, the treatment of helices as a sequence of segments followed by Fourier-Bessel reconstruction revealed the potential to determine near-atomic resolution structures from helical specimens. In the meantime, real-space image processing of helices in a stack of single particles was developed and enabled the structure determination of specimens that resisted classical Fourier helical reconstruction and also facilitated high-resolution structure determination. Despite the progress in real-space analysis, the combination of Fourier and real-space processing is still commonly used to better estimate the symmetry parameters as the imposition of the correct helical symmetry is essential for high-resolution structure determination. Recent hardware advancement by the introduction of direct electron detectors has significantly enhanced the image quality and together with improved image processing procedures has made segmented helical reconstruction a very productive cryo-EM structure determination method.

Virus-Templated Near-Amorphous Iron Oxide Nanotubes.

We present a simple synthesis of iron oxide nanotubes, grown under very mild conditions from a solution containing Fe(II) and Fe(III), on rod-shaped tobacco mosaic virus templates. Their well-defined shape and surface chemistry suggest that these robust bionanoparticles are a versatile platform for synthesis of small, thin mineral tubes, which was achieved efficiently. Various characterization tools were used to explore the iron oxide in detail: Electron microscopy (SEM, TEM), magnetometry (SQUID-VSM), diffraction (XRD, TEM-SAED), electron spectroscopies (EELS, EDX, XPS), and X-ray absorption (XANES with EXAFS analysis). They allowed determination of the structure, crystallinity, magnetic properties, and composition of the tubes. The protein surface of the viral templates was crucial to nucleate iron oxide, exhibiting analogies to biomineralization in natural compartments such as ferritin cages.

Tailoring the Self-Assembly Behaviors of Recombinant Tobacco Mosaic Virus by Rationally Introducing Covalent Bonding at the Protein-Protein Interface.

Understanding the self-assembly mechanism of protein building blocks is important to realize the control of protein structures and functionalities. Here, for the first time, four different self-assembly behaviors of tobacco mosaic virus coat protein are reported from 2D disk arrays, disk stacks to 3D tube stacks, and tube bundles, respectively, with rationally mutated cysteines at 1, 3, and 103 sites.

Benefits and Limitations of Low-kV Macromolecular Imaging of Frozen-Hydrated Biological Samples.

Object contrast is one of the most important parameters of macromolecular imaging. Low-voltage transmission electron microscopy has shown an increased atom contrast for carbon materials, indicating that amplitude contrast contributions increase at a higher rate than phase contrast and inelastic scattering. Here, we studied image contrast using ice-embedded tobacco mosaic virus particles as test samples at 20-80 keV electron energy. The particles showed the expected increase in contrast for lower energies, but at the same time the 2.3-nm-resolution measure decayed more rapidly. We found a pronounced signal loss below 60 keV, and therefore we conclude that increased inelastic scattering counteracts increased amplitude contrast. This model also implies that as long as the amplitude contrast does not increase with resolution, beam damage and multiple scattering will always win over increased contrast at the lowest energies. Therefore, we cannot expect that low-energy imaging of conventionally prepared samples would provide better data than state-of-the-art 200-300 keV imaging.

A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics.

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented.

Multifrequency Force Microscopy of Helical Protein Assembly on a Virus.

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.