From solution to structure: empowering inclusive cryo-EM with a pre-characterization pipeline for biological samples.

In addressing the challenges faced by laboratories and universities with limited (or no) cryo-electron microscopy (cryo-EM) infrastructure, the ESRF, in collaboration with the Grenoble Institute for Structural Biology (IBS), has implemented the cryo-EM Solution-to-Structure (SOS) pipeline. This inclusive process, spanning grid preparation to high-resolution data collection, covers single-particle analysis and cryo-electron tomography (cryo-ET). Accessible through a rolling access route, proposals undergo scientific merit and technical feasibility evaluations. Stringent feasibility criteria demand robust evidence of sample homogeneity. Two distinct entry points are offered: users can either submit purified protein samples for comprehensive processing or initiate the pipeline with already vitrified cryo-EM grids. The SOS pipeline integrates negative stain imaging (exclusive to protein samples) as a first quality step, followed by cryo-EM grid preparation, grid screening and preliminary data collection for single-particle analysis, or only the first two steps for cryo-ET. In both cases, if the screening steps are successfully completed, high-resolution data collection will be carried out using a Titan Krios microscope equipped with a latest-generation direct electron counting detector coupled to an energy filter. The SOS pipeline thus emerges as a comprehensive and efficient solution, further democratizing access to cryo-EM research.

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis.

IMPORTANCE: The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.

Measuring external primary cell wall elasticity of seedling roots using atomic force microscopy.

Stiffness plays a central action in plant cell extension. Here, we present a protocol to detect changes in stiffness on the external epidermal cell wall of living plant roots using atomic force microscopy (AFM). We provide generalized instructions for collecting force-distance curves and analysis of stiffness using contact-based mechanical model. With this protocol, and some initial training in AFM, a user is able to perform indentation experiments on 4- and 5-day-old Arabidopsis thaliana and determine stiffness properties. For complete details on the use and execution of this protocol, please refer to Godon et al.1.

Structure of the human heparan sulfate polymerase complex EXT1-EXT2.

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.

Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba.

RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.

Immediate and Sustained Effects of Cobalt and Zinc-Containing Pigments on Macrophages.

Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt [Pigment Violet 14 (purple color)] or cobalt alloy pigments [Pigment Blue 28 (blue color), Pigment Green 14 (green color)], and one zinc pigment [Pigment White 4 (white color)] which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of submicroparticles or nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in tumor necrosis factor secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced both a short and sustained increase in interleukin 6 secretion. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after exposure to pigments followed by a recovery period. For chemoattractant cytokines (MCP-1 or MIP-1α), delayed effects were observed with a secretion decreased in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional disorders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

Repeated Exposure of Macrophages to Synthetic Amorphous Silica Induces Adaptive Proteome Changes and a Moderate Cell Activation.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

Characterization of a small tRNA-binding protein that interacts with the archaeal proteasome complex.

The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a ß-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.

Structural basis for the inhibition of IAPP fibril formation by the co-chaperonin prefoldin.

Chaperones, as modulators of protein conformational states, are key cellular actors to prevent the accumulation of fibrillar aggregates. Here, we integrated kinetic investigations with structural studies to elucidate how the ubiquitous co-chaperonin prefoldin inhibits diabetes associated islet amyloid polypeptide (IAPP) fibril formation. We demonstrated that both human and archaeal prefoldin interfere similarly with the IAPP fibril elongation and secondary nucleation pathways. Using archaeal prefoldin model, we combined nuclear magnetic resonance spectroscopy with electron microscopy to establish that the inhibition of fibril formation is mediated by the binding of prefoldin's coiled-coil helices to the flexible IAPP N-terminal segment accessible on the fibril surface and fibril ends. Atomic force microscopy demonstrates that binding of prefoldin to IAPP leads to the formation of lower amounts of aggregates, composed of shorter fibrils, clustered together. Linking structural models with observed fibrillation inhibition processes opens perspectives for understanding the interference between natural chaperones and formation of disease-associated amyloids.

Oxygen-Sensitive Metalloprotein Structure Determination by Cryo-Electron Microscopy.

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe4S4]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.

Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects macaques from infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV-2 spike (S) glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter-protomer cross-links that keep all receptor-binding domains in the "down" conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LVs) induces high antibody titers with potent neutralizing activity against the vaccine strain, Alpha, Beta, and Gamma variants as well as T helper (Th)1 CD4+-biased T cell responses. Although anti-receptor-binding domain (RBD)-specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging vaccinated animals with SARS-CoV-2 shows a complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S immunoglobulin G (IgG) and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

The archaeal division protein CdvB1 assembles into polymers that are depolymerized by CdvC.

The Cdv proteins constitute the cell division system of the Crenarchaea, a machinery closely related to the ESCRT system of eukaryotes. Using a combination of TEM imaging and biochemical assays, we here present an in vitro study of Metallosphaera sedula CdvB1, the Cdv protein that is believed to play a major role in the constricting ring that drives cell division in the Crenarchaea. We show that CdvB1 self-assembles into filaments that are depolymerized by the Vps4-homolog ATPase CdvC. Furthermore, we find that CdvB1 binds to negatively charged lipid membranes and can be detached from the membrane by the action of CdvC. Our findings provide novel insight into one of the main components of the archaeal cell division machinery.

Elicitation of potent SARS-CoV-2 neutralizing antibody responses through immunization with a versatile adenovirus-inspired multimerization platform.

Virus-like particles (VLPs) are highly suited platforms for protein-based vaccines. In the present work, we adapted a previously designed non-infectious adenovirus-inspired 60-mer dodecahedric VLP (ADDomer) to display a multimeric array of large antigens through a SpyTag/SpyCatcher system. To validate the platform as a potential COVID-19 vaccine approach, we decorated the newly designed VLP with the glycosylated receptor binding domain (RBD) of SARS-CoV-2. Cryoelectron microscopy structure revealed that up to 60 copies of this antigenic domain could be bound on a single ADDomer particle, with the symmetrical arrangements of a dodecahedron. Mouse immunization with the RBD decorated VLPs already showed a significant specific humoral response following prime vaccination, greatly reinforced by a single boost. Neutralization assays with SARS-CoV-2 spike pseudo-typed virus demonstrated the elicitation of strong neutralization titers, superior to those of COVID-19 convalescent patients. Notably, the presence of pre-existing immunity against the adenoviral-derived particles did not hamper the immune response against the antigen displayed on its surface. This plug and play vaccine platform represents a promising new highly versatile tool to combat emergent pathogens.

About the Transient Effects of Synthetic Amorphous Silica: An In Vitro Study on Macrophages.

Silica (either crystalline or amorphous) is widely used for different applications and its toxicological assessment depends on its characteristics and intended use. As sustained inflammation induced by crystalline silica is at the root of silicosis, investigating the inflammatory effects induced by amorphous silicas and their persistence is needed. For the development of new grades of synthetic amorphous silicas, it is also desirable to be able to understand better the factors underlying potential adverse effects. Therefore, we used an optimized in vitro macrophage system to investigate the effects of amorphous silicas, and their persistence. By using different amorphous silicas, we demonstrated that the main driver for the adverse effects is a low size of the overall particle/agglomerate; the second driver being a low size of the primary particle. We also demonstrated that the effects were transient. By using silicon dosage in cells, we showed that the transient effects are coupled with a decrease of intracellular silicon levels over time after exposure. To further investigate this phenomenon, a mild enzymatic cell lysis allowed us to show that amorphous silicas are degraded in macrophages over time, explaining the decrease in silicon content and thus the transiency of the effects of amorphous silicas on macrophages.

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.

A Low-Serum Culture System for Prolonged in Vitro Toxicology Experiments on a Macrophage System.

Immunotoxicology sensu lato comprises not only toxicity toward immune cells, but also biological reactions from immune cells exposed to toxicants, reactions that may have deleterious effects at the organismal level. Within this wide frame, a specific case of interest is represented by the response of macrophages to particulate materials, with the epitome examples of asbestos and crystalline silica. For such toxicants that are both persistent and often encountered in an occupational setting, i.e. at low but repeated doses, there is a need for in vitro systems that can take into account these two parameters. Currently, most in vitro systems are used in an acute exposure mode, i.e., with a single dose and a readout made shortly if not immediately after exposure. We describe here how adequate changes of the culture methods applied to the murine macrophage cell line J774A.1 enable longer periods of culture (several days), which represents a first opportunity to address the persistence and dose-rate issues. To respond to this, the protocol uses a reduction in the concentration of the animal serum used for cell culture, as well as a switch from fetal to adult serum, which is less rich in proliferation factors. By doing so, we have considerably reduced cell proliferation, which is a problem with cell lines when they are supposed to represent slowly-dividing cells such as resident macrophages. We also succeeded in maintaining the differentiated functions of macrophages, such as phagocytosis or inflammatory responses, over the whole culture period. Furthermore, the presence of serum, even at low concentrations, provides excellent cell viability and keeps the presence of a protein corona on particulate materials, a feature that is known to strongly modulate their effects on cells and is lost in serum-free culture. Besides data showing the impact of these conditions on macrophages cell line cultures, illustrative examples are shown on silica- and cobalt-based pigments.

How Reversible Are the Effects of Fumed Silica on Macrophages? A Proteomics-Informed View.

Synthetic amorphous silica is one of the most used nanomaterials, and numerous toxicological studies have studied its effects. Most of these studies have used an acute exposure mode to investigate the effects immediately after exposure. However, this exposure modality does not allow the investigation of the persistence of the effects, which is a crucial aspect of silica toxicology, as exemplified by crystalline silica. In this paper, we extended the investigations by studying not only the responses immediately after exposure but also after a 72 h post-exposure recovery phase. We used a pyrolytic silica as the test nanomaterial, as this variant of synthetic amorphous silica has been shown to induce a more persistent inflammation in vivo than precipitated silica. To investigate macrophage responses to pyrolytic silica, we used a combination of proteomics and targeted experiments, which allowed us to show that most of the cellular functions that were altered immediately after exposure to pyrolytic silica at a subtoxic dose, such as energy metabolism and cell morphology, returned to normal at the end of the recovery period. However, some alterations, such as the inflammatory responses and some aldehyde detoxification proteins, were persistent. At the proteomic level, other alterations, such as proteins implicated in the endosomal/lysosomal pathway, were also persistent but resulted in normal function, thus suggesting cellular adaptation.

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2.

The study of viruses causing acute respiratory distress syndromes (ARDS) is more essential than ever at a time when a virus can create a global pandemic in a matter of weeks. Among human adenoviruses, adenovirus of serotype 7 (HAdV7) is one of the most virulent serotypes. This virus regularly re-emerges in Asia and has just been the cause of several deaths in the United States. A critical step of the virus life cycle is the attachment of the knob domain of the fiber (HAd7K) to the cellular receptor desmoglein-2 (DSG2). Complexes between the fiber knob and two extracellular domains of DSG2 have been produced. Their characterization by biochemical and biophysical methods show that these two domains are sufficient for the interaction and that the trimeric HAd7K could accommodate up to three DSG2 receptor molecules. The cryo-electron microscopy (cryo-EM) structure of these complexes at 3.1 Å resolution confirmed the biochemical data, and allowed the identification of the critical amino acid residues for this interaction, which shows similarities with other DSG2 interacting adenoviruses, despite a low homology in the primary sequences.