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Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses., Competing Interests: Conflict of interest D.A.K.T. is an employee of Thermo Fisher Scientific, a company dedicated to advancing electron cryo-imaging technology. Other authors declare no conflict of interest.

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Conjugative DNA transfer is a major factor in the dissemination of antibiotic resistance and virulence genes. In the Gram-positive pathogen Clostridium perfringens, the majority of conjugative plasmids share the conserved tcp locus that governs the assembly of the transfer system. Here, we describe multiple structures of the coupling protein TcpA, an essential ATPase that is suggested to provide the mechanical force to propel the DNA through the transfer apparatus. The structures of TcpA in the presence and absence of nucleotides revealed conformational rearrangements and highlight a crucial role for the unstructured C terminus. Our findings reveal that TcpA shares most structural similarity with the FtsK DNA translocase, a central component of the bacterial cell division machinery. Our structural data suggest that conjugation in C. perfringens may have evolved from the bacterial chromosome segregation system and, accordingly, suggest the possibility that double-stranded DNA is transferred through the Tcp conjugation apparatus., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Africa is the only habitable continent that is not yet host to a light source - an important tool across disciplines. Scientists from the Executive Committee of the African Light Source Foundation discuss work towards building an advanced light source in Africa, and what remains to be done., Competing Interests: Competing interestsThe authors declare no competing interests., (© Springer Nature Limited 2022.)

In this work, the inclusion complexes of alkyl ethoxy carboxylates with α-cyclodextrin (αCD) and β-cyclodextrin (βCD) were investigated. The thermodynamics of the complexation process was probed by isothermal titration calorimetry (ITC) and volumetry as a function of the degree of ionization of the surfactant. The complexation process was shown to be an enthalpically driven pH-independent process. For both types of cyclodextrins, the complexes were found to spontaneously self-assemble into highly-ordered supramolecular aggregates probed by small-angle neutron scattering and electron and optical microscopy. Herein, we report the formation of thin platelets for nonionized surfactant systems and equally spaced multilayered hollow cylinders for ionized systems in a hierarchical self-assembly process. In addition, the analysis allowed unveiling the effect of the number of ethylene oxides in the surfactants and the CD cavity size on the morphology of the aggregates. Finally, this study also highlights the importance of examining the tuning parameters' influence on the short and long-range interactions involved in the control of the assembly process.

Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system; in C. perfringens plasmids, there are approximately 10 different ParMRC families, with significant differences in amino acid sequences between each ParM family (15% to 54% identity). Since plasmids carrying genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility in C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site from the same ParMRC family but could not interact with a parC site from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC -like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets. IMPORTANCE Toxins produced by the Gram-positive pathogen Clostridium perfringens are primarily encoded by genes found on different conjugative plasmids. These plasmids encode highly similar replication proteins and therefore should be incompatible, but they are often found to coexist within the same isolate. In this study, we showed that a series of phylogenetically related ParMRC plasmid partitioning systems, structures that are normally responsible for ensuring that plasmids segregate correctly at cell division, dictate which toxin plasmid combinations can coexist within the same bacterial cell. We dissected the recognition steps between the DNA-binding ParMRC component, ParR, and the plasmid-derived centromere, parC . Our data suggested a mechanism by which plasmids encoding ParMRC systems from the same family are incompatible, whereas plasmids encoding ParMRC systems from distinct families are compatible. This work provides insight into how these cells can maintain multiple highly similar toxin plasmids, which is a critical first step in understanding how to limit the disease-causing potential of C. perfringens.

Structural biology is an essential tool for understanding the molecular basis of diseases, which can guide the rational design of new drugs, vaccines, and the optimisation of existing medicines. However, most African countries do not conduct structural biology research due to limited resources, lack of trained persons, and an exodus of skilled scientists. The most urgent requirement is to build on the emerging centres in Africa - some well-established, others growing. This can be achieved through workshops that improve networking, grow skills, and develop mechanisms for access to light source beamlines for defining X-ray structures across the continent. These would encourage the growth of structural biology, which is central to understanding biological functions and developing new antimicrobials and other drugs. In this light, a hands-on training workshop in structural biology series 4 was organised by BioStruct-Africa and the Malaria Research and Training Center (MRTC) in Bamako, Mali, to help bridge this gap. The workshop was hosted by MRTC from the 25th to 28th of April 2022. Through a series of lectures and practicals, the workshop enlightened the participants on how structural biology can be utilised to find solutions to the prevalent diseases in Africa. The short training gave them an overview of target selection, protein production and purification, structural determination techniques, and analysis in combination with high-throughput, structure-guided, fragment-based drug design., Competing Interests: Competing interests D.A.K.T. is the co-founder, and E.N. is the CEO and co-founder of BioStruct-Africa. Along with the co-authors, they declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The majority of existing studies aimed at investigating the incidence and prevalence of multidrug-resistance by bacteria have been performed in healthcare settings. Relatively few studies have been conducted in community settings, but these have consistently shown a high prevalence of multidrug-resistant bacteria in low- and middle-income countries (LMICs)., Objectives: To provide an appraisal of the evidence on the high prevalence of multidrug-, extensive drug-, and pandrug-resistance in commensal Escherichia coli isolates from human sources in community settings in LMICs., Methods: Using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines, PubMed, EMBASE, MEDLINE, Web of Science, CINAHL, and Cochrane Library databases were systematically searched with the search string: 'Enterobacteriaceae', OR ' E. coli ', OR ' Escherichia coli ', AND 'antibiotic resistance', OR 'antimicrobial resistance', OR 'drug-resistance', AND 'prevalence', OR 'incidence', OR 'morbidity', OR 'odds ratio', OR 'risk ratio', OR 'confidence interval', OR 'p-value', OR 'rate'. Data were extracted and proportional meta-analysis was performed using the Freeman-Tukey transformation random effect model., Results: The prevalence of multidrug-, extensive drug- and pandrug-resistance were extracted from articles that met our inclusion criteria and pooled together after a systematic screening of 9,369 items. The prevalence of multidrug-resistance was 28% of 14,336 total cases of isolates tested, 95% CI: 23-32. Extensive drug-resistance was 24% of 8,686 total cases of isolates tested, 95% CI: 14-36. Lastly, pandrug-resistance was 5% of 5,670 total cases of isolates tested, 95% CI: 3-8., Conclusion: This paper provides an appraisal of the evidence on the high prevalence of multidrug-, extensive drug- and pandrug-resistance by commensal E. coli in community settings in LMICs. Our results call for greater effort to be placed at the community level in the design of new and improved public health policies to counter the global threat of antibiotic-resistant infections and bacteria.

Being able to visualize biology at the molecular level is essential for our understanding of the world. A structural biology approach reveals the molecular basis of disease processes and can guide the design of new drugs as well as aid in the optimization of existing medicines. However, due to the lack of a synchrotron light source, adequate infrastructure, skilled persons and incentives for scientists in addition to limited financial support, the majority of countries across the African continent do not conduct structural biology research. Nevertheless, with technological advances such as robotic protein crystallization and remote data collection capabilities offered by many synchrotron light sources, X-ray crystallography is now potentially accessible to Africa-based scientists. This leap in technology led to the establishment in 2017 of BioStruct-Africa, a non-profit organization (Swedish corporate ID: 802509-6689) whose core aim is capacity building for African students and researchers in the field of structural biology with a focus on prevalent diseases in the African continent. The team is mainly composed of, but not limited to, a group of structural biologists from the African diaspora. The members of BioStruct-Africa have taken up the mantle to serve as a catalyst in order to facilitate the information and technology transfer to those with the greatest desire and need within Africa. BioStruct-Africa achieves this by organizing workshops onsite at our partner universities and institutions based in Africa, followed by post-hoc online mentoring of participants to ensure sustainable capacity building. The workshops provide a theoretical background on protein crystallography, hands-on practical experience in protein crystallization, crystal harvesting and cryo-cooling, live remote data collection on a synchrotron beamline, but most importantly the links to drive further collaboration through research. Capacity building for Africa-based researchers in structural biology is crucial to win the fight against the neglected tropical diseases, e.g. ascariasis, hookworm, trichuriasis, lymphatic filariasis, active trachoma, loiasis, yellow fever, leprosy, rabies, sleeping sickness, onchocerciasis, schistosomiasis, etc., that constitute significant health, social and economic burdens to the continent. BioStruct-Africa aims to build local and national expertise that will have direct benefits for healthcare within the continent.

Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK-TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.

Macrophage migration inhibitory factor (MIF) exerts multiple effects on immune cells, as well as having functions outside the immune system. MIF can promote inflammation through the induction of other cytokines, including TNF, IL-6, and IL-1 family cytokines. Here, we show that inhibition of MIF regulates the release of IL-1α, IL-1β, and IL-18, not by affecting transcription or translation of these cytokines, but via activation of the NLRP3 inflammasome. MIF is required for the interaction between NLRP3 and the intermediate filament protein vimentin, which is critical for NLRP3 activation. Further, we demonstrate that MIF interacts with NLRP3, indicating a role for MIF in inflammasome activation independent of its role as a cytokine. These data advance our understanding of how MIF regulates inflammation and identify it as a factor critical for NLRP3 inflammasome activation.