A stargate mechanism of Microviridae genome delivery unveiled by cryogenic electron tomography.

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.

Structural conservation of insulin/IGF signalling axis at the insulin receptors level in Drosophila and humans.

The insulin-related hormones regulate key life processes in Metazoa, from metabolism to growth, lifespan and aging, through an evolutionarily conserved insulin signalling axis (IIS). In humans the IIS axis is controlled by insulin, two insulin-like growth factors, two isoforms of the insulin receptor (hIR-A and -B), and its homologous IGF-1R. In Drosophila, this signalling engages seven insulin-like hormones (DILP1-7) and a single receptor (dmIR). This report describes the cryoEM structure of the dmIR ectodomain:DILP5 complex, revealing high structural homology between dmIR and hIR. The excess of DILP5 yields dmIR complex in an asymmetric 'T' conformation, similar to that observed in some complexes of human IRs. However, dmIR binds three DILP5 molecules in a distinct arrangement, showing also dmIR-specific features. This work adds structural support to evolutionary conservation of the IIS axis at the IR level, and also underpins a better understanding of an important model organism.

In crystallo lattice adaptivity triggered by solid-gas reactions of cationic group 7 pincer complexes.

The group 7 complexes [M(κ3-2,6-(R2PO)2C5H3N)(CO)2L][BArF4] [M = Mn, R = iPr, L = THF; M = Re, R = tBu, L = vacant site] undergo in crystallo solid-gas reactivity with CO to form the products of THF substitution or CO addition respectively. There is a large, local, adaptive change of [BArF4] anions for M = Mn, whereas for M = Re the changes are smaller and also remote to the site of reactivity.

Structure of HK97 small terminase:DNA complex unveils a novel DNA binding mechanism by a circular protein.

DNA recognition is critical for assembly of double-stranded DNA viruses, in particular for the initiation of packaging the viral genome into the capsid. DNA packaging has been extensively studied for three archetypal bacteriophage systems: cos, pac and phi29. We identified the minimal site within the cos region of bacteriophage HK97 specifically recognised by the small terminase and determined a cryoEM structure for the small terminase:DNA complex. This nonameric circular protein utilizes a previously unknown mechanism of DNA binding. While DNA threads through the central tunnel, unexpectedly, DNA-recognition is generated at its exit by a substructure formed by the N- and C-terminal segments of two adjacent protomers of the terminase which are unstructured in the absence of DNA. Such interaction ensures continuous engagement of the small terminase with DNA, allowing sliding along DNA while simultaneously checking the DNA sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the cos site.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

An observational investigation of the faical microbiota and metabonome of gastrostomy fed children, on blended and formula diets.

Gastrostomy fed children traditionally have a Formulae diet (FD), which fulfills nutritional requirements; however, many families are adopting Blended diets (BD), which are what the whole family would eat. We undertook an observational investigation of the colonic microbiota and metabonome in a small group of gastrostomy fed children, who were either on an FD or BD, and compared, where possible to their siblings (17 FD, 28 BD, 19 HS). There was no increase in complications in tube blockage or infection rates, but a significant improvement in the prevalence of bowel problems, a reduction in medication and an increase in quality of life. Metataxonomic analysis showed that the FD group was significantly different to the Sibling group, and that families did not cluster together. Whole sample metabonomics showed no differences between groups; however, univariate analysis of biologically important metabolites did differ. Changing to a BD resulted in no increase in complications or risks, but improved the overall quality of life for the children and families.

MicroED characterization of a robust cationic σ-alkane complex stabilized by the [B(3,5-(SF5)2C6H3)4]- anion, via on-grid solid/gas single-crystal to single-crystal reactivity.

Microcrystalline (∼1 µm) [Rh(Cy2PCH2CH2PCy2)(norbornadiene)][S-BArF4], [S-BArF4] = [B(3,5-(SF5)2C6H3)4]-, reacts with H2 in a single-crystal to single-crystal transformation to form the σ-alkane complex [Rh(Cy2PCH2CH2PCy2)(norbornane)][S-BArF4], for which the structure was determined by microcrystal Electron Diffraction (microED), to 0.95 Å resolution, via an on-grid hydrogenation, and a complementary single-crystal X-ray diffraction study on larger, but challenging to isolate, crystals. Comparison with the [BArF4]- analogue [ArF = 3,5-(CF3)2(C6H3)] shows that the [S-BArF4]- anion makes the σ-alkane complex robust towards decomposition both thermally and when suspended in pentane. Subsequent reactivity with dissolved ethene in a pentane slurry, forms [Rh(Cy2PCH2CH2PCy2)(ethene)2][S-BArF4], and the catalytic dimerisation/isomerisation of ethene to 2-butenes. The increased stability of [S-BArF4]- salts is identified as being due to increased non-covalent interactions in the lattice, resulting in a solid-state molecular organometallic material with desirable stability characteristics.

CryoEM structure of the Nipah virus nucleocapsid assembly.

Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the "3-bases-in, 3-bases-out" conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123-139, which may serve as a valuable epitope for surveillance and diagnostics.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor.

Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3 - and AlF4 -, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)-, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)- species, indicating the significance of this TSA for studies of biological motors.

Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase.

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an "AT3-SGNH fused" domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates.

Photoreceptor Activity Contributes to Contrasting Responses to Shade in Cardamine and Arabidopsis Seedlings.

Plants have evolved two major ways to deal with nearby vegetation or shade: avoidance and tolerance. Moreover, some plants respond to shade in different ways; for example, Arabidopsis (Arabidopsis thaliana) undergoes an avoidance response to shade produced by vegetation, but its close relative Cardamine hirsuta tolerates shade. How plants adopt opposite strategies to respond to the same environmental challenge is unknown. Here, using a genetic strategy, we identified the C. hirsuta slender in shade1 mutants, which produce strongly elongated hypocotyls in response to shade. These mutants lack the phytochrome A (phyA) photoreceptor. Our findings suggest that C. hirsuta has evolved a highly efficient phyA-dependent pathway that suppresses hypocotyl elongation when challenged by shade from nearby vegetation. This suppression relies, at least in part, on stronger phyA activity in C. hirsuta; this is achieved by increased ChPHYA expression and protein accumulation combined with a stronger specific intrinsic repressor activity. We suggest that modulation of photoreceptor activity is a powerful mechanism in nature to achieve physiological variation (shade tolerance versus avoidance) for species to colonize different habitats.

Fragon: rapid high-resolution structure determination from ideal protein fragments.

Correctly positioning ideal protein fragments by molecular replacement presents an attractive method for obtaining preliminary phases when no template structure for molecular replacement is available. This has been exploited in several existing pipelines. This paper presents a new pipeline, named Fragon, in which fragments (ideal α-helices or ß-strands) are placed using Phaser and the phases calculated from these coordinates are then improved by the density-modification methods provided by ACORN. The reliable scoring algorithm provided by ACORN identifies success. In these cases, the resulting phases are usually of sufficient quality to enable automated model building of the entire structure. Fragon was evaluated against two test sets comprising mixed α/ß folds and all-ß folds at resolutions between 1.0 and 1.7 Å. Success rates of 61% for the mixed α/ß test set and 30% for the all-ß test set were achieved. In almost 70% of successful runs, fragment placement and density modification took less than 30 min on relatively modest four-core desktop computers. In all successful runs the best set of phases enabled automated model building with ARP/wARP to complete the structure.

CCP4i2: the new graphical user interface to the CCP4 program suite.

The CCP4 (Collaborative Computational Project, Number 4) software suite for macromolecular structure determination by X-ray crystallography groups brings together many programs and libraries that, by means of well established conventions, interoperate effectively without adhering to strict design guidelines. Because of this inherent flexibility, users are often presented with diverse, even divergent, choices for solving every type of problem. Recently, CCP4 introduced CCP4i2, a modern graphical interface designed to help structural biologists to navigate the process of structure determination, with an emphasis on pipelining and the streamlined presentation of results. In addition, CCP4i2 provides a framework for writing structure-solution scripts that can be built up incrementally to create increasingly automatic procedures.

Real-life Anti-tumor Necrosis Factor Experience in More Than 500 Patients: High Co-immunosuppression Rates But Low Rates of Quantifying Treatment Response.

OBJECTIVE: The aim of this study was to measure the effectiveness, safety, and use of anti-tumor necrosis Factor (TNF) therapy in pediatric inflammatory bowel disease in the United Kingdom (UK). METHODS: Prospective UK audit of patients newly starting anti-TNF therapy. Disease severity was assessed using Physician Global Assessment +/or the Paediatric Crohn Disease Activity Index. RESULTS: A total of 37 centers participated (23/25 specialist pediatric inflammatory bowel disease sites). A total of 524 patients were included: 429 with Crohn disease (CD), 76 with ulcerative colitis (UC), and 19 with IBD unclassified (IBDU). Eighty-seven percent (488/562) of anti-TNF was infliximab; commonest indication was active luminal CD 77% (330/429) or chronic refractory UC/IBDU 56% (53/95); 79% (445/562) had concomitant co-immunosuppression. In CD (267/429 male), median time from diagnosis to treatment was 1.42 years (interquartile range 0.63-2.97). Disease (at initiation) was moderate or severe in 91% (156/171) by Physician Global Assessment compared to 41% (88/217) by Paediatric Crohn Disease Activity Index (Kappa (κ) 0.28 = only "fair agreement"; P < 0.001.Where documented, 77% (53/69) of patients with CD responded to induction; and 65% (46/71) entered remission. A total of 2287 infusions and 301.96 years of patient' follow-up (n = 385) are represented; adverse events affected 3% (49/1587) infliximab and 2% (2/98) adalimumab infusions (no deaths or malignancies). Peri-anal abscess drainage was less common after anti-TNF initiation (CD), that is 26% (27/102) before, 7% (3/42) after (P = 0.01); however, pre and post anti-TNF data collection was not over equal time periods. CONCLUSIONS: Anti-TNFs are effective treatments, usually given with thiopurine co-immunosuppression. This study highlights deficiencies in formal documentation of effect and disparity between disease severity scoring tools, which need to be addressed to improve ongoing patient care.

Automating tasks in protein structure determination with the clipper python module.

Scripting programming languages provide the fastest means of prototyping complex functionality. Those with a syntax and grammar resembling human language also greatly enhance the maintainability of the produced source code. Furthermore, the combination of a powerful, machine-independent scripting language with binary libraries tailored for each computer architecture allows programs to break free from the tight boundaries of efficiency traditionally associated with scripts. In the present work, we describe how an efficient C++ crystallographic library such as Clipper can be wrapped, adapted and generalized for use in both crystallographic and electron cryo-microscopy applications, scripted with the Python language. We shall also place an emphasis on best practices in automation, illustrating how this can be achieved with this new Python module.

Social and health outcomes following upgrades to a national housing standard: a multilevel analysis of a five-wave repeated cross-sectional survey.

BACKGROUND: While existing research indicates that housing improvements are associated with health improvements, less is known about the wider social and health benefits of meeting national housing standards, as well as those of their specific constituent measures. This study evaluates the impacts of a managed housing upgrade programme through a repeated cross-sectional survey design. METHODS: A five-wave repeated cross-sectional survey was conducted over a seven-year period from 2009 to 2016 (n = 2075; n = 2219; n = 2015; n = 1991; and n = 1709, respectively). The study followed a managed upgrade programme designed to meet a national social housing standard over an extended period. The data were analysed from a multilevel perspective to take account of the time-dependent nature of the observations and differences in socio-demographic composition. RESULTS: The installation of the majority of individual housing measures (new windows and doors; boilers; kitchens; bathrooms; electrics; loft insulation; and cavity/external wall insulation) were associated with improvements in several social (housing suitability, satisfaction, and quality; thermal comfort and household finances) and health (mental, respiratory and general health) outcomes; and analyses showed relationships between the number of measures installed and the total amount invested on the one hand and the social and health outcomes on the other. There were however a few exceptions. Most notably, the installation of cavity wall insulation was associated with poorer health outcomes, and did not lead to better social outcomes. Also, no association was found between the number of measures installed and respiratory health. CONCLUSIONS: The study suggests that substantial housing investments through a managed upgrade programme may result in better social and health outcomes, and that the size of the improvements are proportionate to the number of measures installed and amount invested. However, there may be risks associated with specific measures; and more attention is needed for mechanical ventilation when upgrading energy efficiency of houses through fabric work. In addition to providing new evidence regarding the wider social and health outcomes, the study provides an analytical approach to evaluate upgrade programmes that are delivered over multiple years.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.