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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., (© Christoph Mueller-Dieckmann et al. 2024.)

Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms., (© 2023. The Author(s).)

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum . They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda ( =kdgA ) , gudD, garK, garL , and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients ( dpp , ddp , dctA , and pst ) including glucuronate ( kdgK and yeiQ ) and synthesis of metabolites: cellulose ( celY and bcs ), aryl polyene ( ape ), and oocydin ( ooc ). We constructed in-frame deletion mutants of bcsA, ddpA, apeH , and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the Δ pstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Robic, Munier, Effantin, Lachat, Naquin, Gueguen and Faure.)

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Brual et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacteriophages, viruses infecting bacteria, recognize their host with high specificity, binding to either saccharide motifs or proteins of the cell wall of their host. In the majority of bacteriophages, this host recognition is performed by receptor binding proteins (RBPs) located at the extremity of a tail. Interaction between the RBPs and the host is the trigger for bacteriophage infection, but the molecular details of the mechanisms are unknown for most bacteriophages. Here, we present the electron cryomicroscopy (cryo-EM) structure of bacteriophage T5 RBP pb5 in complex with its Escherichia coli receptor, the iron ferrichrome transporter FhuA. Monomeric RBP pb5 is located at the extremity of T5's long flexible tail, and its irreversible binding to FhuA commits T5 to infection. Analysis of the structure of RBP pb5 within the complex, comparison with its AlphaFold2-predicted structure, and its fit into a previously determined map of the T5 tail tip in complex with FhuA allow us to propose a mechanism of transmission of the RBP pb5 receptor binding to the straight fiber, initiating the cascade of events that commits T5 to DNA ejection. IMPORTANCE Tailed bacteriophages specifically recognize their bacterial host by interaction of their receptor binding protein(s) (RBPs) with saccharides and/or proteins located at the surface of their prey. This crucial interaction commits the virus to infection, but the molecular details of this mechanism are unknown for the majority of bacteriophages. We determined the structure of bacteriophage T5 RBP pb5 in complex with its E. coli receptor, FhuA, by cryo-EM. This first structure of an RBP bound to its protein receptor allowed us to propose a mechanism of transmission of host recognition to the rest of the phage, ultimately opening the capsid and perforating the cell wall and, thus, allowing safe channeling of the DNA into the host cytoplasm.

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail ( Siphoviridae ) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.

The SARS-CoV-2 pandemic has again shown that structural biology plays an important role in understanding biological mechanisms and exploiting structural data for therapeutic interventions. Notably, previous work on SARS-related glycoproteins has paved the way for the rapid structural determination of the SARS-CoV-2 S glycoprotein, which is the main target for neutralizing antibodies. Therefore, all vaccine approaches aimed to employ S as an immunogen to induce neutralizing antibodies. Like all enveloped virus glycoproteins, SARS-CoV-2 S native prefusion trimers are in a metastable conformation, which primes the glycoprotein for the entry process via membrane fusion. S-mediated entry is associated with major conformational changes in S, which can expose many off-target epitopes that deviate vaccination approaches from the major aim of inducing neutralizing antibodies, which mainly target the native prefusion trimer conformation. Here, we review the viral glycoprotein stabilization methods developed prior to SARS-CoV-2, and applied to SARS-CoV-2 S, in order to stabilize S in the prefusion conformation. The importance of structure-based approaches is highlighted by the benefits of employing stabilized S trimers versus non-stabilized S in vaccines with respect to their protective efficacy.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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.