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The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

Beamline B21 at the Diamond Light Source synchrotron in the UK is a small‐angle X‐ray scattering (SAXS) beamline that specializes in high‐throughput measurements via automated sample delivery systems. A system has been developed whereby a sample can be illuminated by a focused beam of light coincident with the X‐ray beam. The system is compatible with the highly automated sample delivery system at the beamline and allows a beamline user to select a light source from a broad range of wavelengths across the UV and visible spectrum and to control the timing and duration of the light pulse with respect to the X‐ray exposure of the SAXS measurement. The intensity of the light source has been characterized across the wavelength range enabling experiments where a quantitative measure of dose is important. Finally, the utility of the system is demonstrated via measurement of several light‐responsive samples. [ABSTRACT FROM AUTHOR]

Plasminogen (Plg) is the inactive form of plasmin (Plm) that exists in two major glycoforms, referred to as glycoforms I and II (GI and GII). In the circulation, Plg assumes an activation-resistant "closed" conformation via interdomain interactions and is mediated by the lysine binding site (LBS) on the kringle (KR) domains. These inter-domain interactions can be readily disrupted when Plg binds to lysine/arginine residues on protein targets or free L-lysine and analogues. This causes Plg to convert into an "open" form, which is crucial for activation by host activators. In this study, we investigated how various ligands affect the kinetics of Plg conformational change using small-angle X-ray scattering (SAXS). We began by examining the open and closed conformations of Plg using size-exclusion chromatography (SEC) coupled with SAXS. Next, we developed a high-throughput (HTP) 96-well SAXS assay to study the conformational change of Plg. This method enables us to determine the Kopen value, which is used to directly compare the effect of different ligands on Plg conformation. Based on our analysis using Plg GII, we have found that the Kopen of ε-aminocaproic acid (EACA) is approximately three times greater than that of tranexamic acid (TXA), which is widely recognized as a highly effective ligand. We demonstrated further that Plg undergoes a conformational change when it binds to the C-terminal peptides of the inhibitor α2-antiplasmin (α2AP) and receptor Plg–RKT. Our findings suggest that in addition to the C-terminal lysine, internal lysine(s) are also necessary for the formation of open Plg. Finally, we compared the conformational changes of Plg GI and GII directly and found that the closed form of GI, which has an N-linked glycosylation, is less stable. To summarize, we have successfully determined the response of Plg to various ligand/receptor peptides by directly measuring the kinetics of its conformational changes. [ABSTRACT FROM AUTHOR]

The current SARS-Covid-2 (SARS-CoV-2) pandemic has led to an acceleration of messenger ribonucleic acid (mRNA) vaccine technology. The development of production processes for these large mRNA molecules, especially self-amplifying mRNA (saRNA), has required concomitant development of analytical characterization techniques. Characterizing the purity, shape and structure of these biomolecules is key to their successful performance as drug products. This article describes the biophysical characterization of the Imperial College London Self-amplifying viral RNA vaccine (IMP-1) developed for SARS-CoV-2. A variety of analytical techniques have been used to characterize the IMP-1 RNA molecule. In this article, we use ultraviolet spectroscopy, dynamic light scattering, size-exclusion chromatography small-angle X-ray scattering and circular dichroism to determine key biophysical attributes of IMP-1. Each technique provides important information about the concentration, size, shape, structure and purity of the molecule. [ABSTRACT FROM AUTHOR]

Nanofibres are an interesting phase into which amphiphilic molecules can self-assemble. Described for a large number of synthetic lipids, they were seldom reported for natural lipids like microbial amphiphiles, known as biosurfactants. In this work, we show that the palmitic acid congener of sophorolipids (SLC16:0), one of the most studied families of biosurfactants, spontaneously forms a self-assembled fibre network (SAFiN) at pH below 6 through a pH jump process. pH-resolved in situ small-angle X-ray scattering (SAXS) shows a continuous micelle-to-fibre transition, characterized by an enhanced core–shell contrast between pH 9 and pH 7 and micellar fusion into a flat membrane between pH 7 and pH 6, approximately. Below pH 6, homogeneous, infinitely long nanofibres form by peeling off the membranes. Eventually, the nanofibre network spontaneously forms a thixotropic hydrogel with fast recovery rates after applying an oscillatory strain amplitude out of the linear viscoelastic regime: after being submitted to strain amplitudes during 5 min, the hydrogel recovers about 80% and 100% of its initial elastic modulus after, respectively, 20 s and 10 min. Finally, the strength of the hydrogel depends on the medium's final pH, with an elastic modulus fivefold higher at pH 3 than at pH 6. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'. [ABSTRACT FROM AUTHOR]

We show how to control the formation and alignment of gel 'noodles'. Nanostructure alignment can be achieved reproducibly by extensional deformation as the filaments form. Using a spinning technique, very long and highly aligned filaments can be made. The Young's moduli of the gel noodles are similar to that of a bulk gel. By using two syringe pumps in a concentric flow setup, we show that a filament-in-filament morphology can be created. [ABSTRACT FROM AUTHOR]

The design of a multipurpose sample cell holder for the high‐throughput (HT) beamline B21 is presented. The device is compatible with the robot bioSAXS sample changer currently installed on BM29, ESRF, and P12 Petra IV synchrotrons. This work presents an approach that uses 3D‐printing to make hardware alterations which can expand the versatility of HT beamlines at low cost. [ABSTRACT FROM AUTHOR]

Amino acid functionalized perylene bisimides (PBIs) form self‐assembled structures in solution, the nature of which depends on the local environment. Using a high‐throughput photocatalysis setup, five PBIs are studied for the hydrogen evolution reaction (HER) under a range of conditions (pH and hole scavenger concentration) across 350 experiments to explore the relationship between supramolecular structure and photocatalytic activity. Using small angle X‐ray scattering (SAXS), NMR spectroscopy and ultraviolet‐visible (UV‐vis) absorption spectroscopy, it is shown that photocatalytic activity is determined by the nature of the self‐assembled aggregate that is formed, demonstrating the potential of self‐assembly to tune activity. There is a clear correlation between the presence of charged flexible cylindrical aggregates and the occurrence of photocatalytic H2 production, with UV–vis spectroscopy indicating that the most active structure type has a distinctive form of π‐aggregation which is proposed to enable efficient charge separation across multiple PBI units. [ABSTRACT FROM AUTHOR]

B21 is a small‐angle X‐ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi‐layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in‐vacuum Eiger 4M (Dectris) detector. A high‐performance liquid chromatography system and a liquid‐handling robot make it possible to load solution samples into a temperature‐controlled in‐vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å−1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013. [ABSTRACT FROM AUTHOR]

Exceptionally large crystals of posnjakite, Cu4SO4(OH)6(H2O), formed during corrosion of a Swagelock(tm) Snubber copper gasket within the MX1 beamline at the ANSTO-Melbourne, Australian Synchrotron. The crystal structure was solved using synchrotron radiation to R1 = 0.029 and revealed a structure based upon [Cu4(OH)6(H2O)O] sheets, which contain Jahn–Teller-distorted Cu octa­hedra. The sulfate tetrahedra are bonded to one side of the sheet via corner sharing and linked to successive sheets via extensive hydrogen bonds. The sulfate tetra­hedra are split and rotated, which enables additional hydrogen bonds. [ABSTRACT FROM AUTHOR]

The micellar aggregates formed at high pH for dipeptide-based gelators can be varied by using different alkali metal salts to prepare the solutions. The nature of the micellar aggregates directly affects the properties of the resulting gels. [ABSTRACT FROM AUTHOR]

Non-invasive manipulation of the hierarchical structure of functional materials is a key challenge in the advancement of optoelectronics, energy conversion and storage devices and drug delivery systems. Lyotropic liquids crystals, with their long-range order and a rich array of structures, have long been exploited as soft templates to prepare porous materials. The potential to modify the template structure using an external stimulus such as light could lead to new architectures. Here, using a combination of small-angle X-ray scattering and polarised optical microscopy, we decipher the various structure/self-assembly relationships of neutral surfactants bearing photoswitchable tails, which self-organise into a rich variety of lyotropic liquid crystalline mesophases. Facile, multimodal control of the nanoscale morphology of these single-component systems is achieved through: (i) molecular structure, via careful selection of the alkyl tail/ethylene oxide headgroup lengths; (ii) concentration; (iii) temperature; and (iv) photoisomerisation. The nanoscale architectures range from the weakly concentrated hyperswollen lamellar phases, the more common lyotropic lamellar and hexagonal phases, to pure thermotropic liquid crystals; all of which are accessible at room temperature. Photoisomerisation with UV light leads to the reversible destruction of the liquid crystalline phase, which can be spatially controlled through the use of a mask. This extensive study demonstrates the versatility of neutral photosurfactants and paves the way for new applications, such as photoresponsive templates or drug delivery systems. [ABSTRACT FROM AUTHOR]

MX2 is an in‐vacuum undulator‐based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X‐rays in the energy range 4.8–21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s−1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard‐to‐crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights. [ABSTRACT FROM AUTHOR]

Radiation damage is a major limitation to synchrotron small-angle X-ray scattering analysis of biomacromolecules. Flowing the sample during exposure helps to reduce the problem, but its effectiveness in the laminar-flow regime is limited by slow flow velocity at the walls of sample cells. To overcome this limitation, the coflow method was developed, where the sample flows through the centre of its cell surrounded by a flow of matched buffer. The method permits an order-of-magnitude increase of X-ray incident flux before sample damage, improves measurement statistics and maintains low sample concentration limits. The method also efficiently handles sample volumes of a few microlitres, can increase sample throughput, is intrinsically resistant to capillary fouling by sample and is suited to static samples and size-exclusion chromatography applications. The method unlocks further potential of third-generation synchrotron beamlines to facilitate new and challenging applications in solution scattering. [ABSTRACT FROM AUTHOR]

The growing problem of antibiotic resistance underlies the critical need to develop new treatments to prevent and control resistant bacterial infection. Exogenous application of bacteriophage lysins results in rapid and specific destruction of Gram-positive bacteria and therefore lysins represent novel antibacterial agents. The PlyC phage lysin is the most potent lysin characterized to date and can rapidly lyse Group A, C and E streptococci. Previously, we have determined the X-ray crystal structure of PlyC, revealing a complicated and unique arrangement of nine proteins. The scaffold features a multimeric cell-wall docking assembly bound to two catalytic domains that communicate and work synergistically. However, the crystal structure appeared to be auto-inhibited and raised important questions as to the mechanism underlying its extreme potency. Here we use small angle X-ray scattering (SAXS) and reveal that the conformational ensemble of PlyC in solution is different to that in the crystal structure. We also investigated the flexibility of the enzyme using both normal mode (NM) analysis and molecular dynamics (MD) simulations. Consistent with our SAXS data, MD simulations show rotational dynamics of both catalytic domains, and implicate inter-domain communication in achieving a substrate-ready conformation required for enzyme function. Our studies therefore provide insights into how the domains in the PlyC holoenzyme may act together to achieve its extraordinary potency. [ABSTRACT FROM AUTHOR]

MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented. [ABSTRACT FROM AUTHOR]

Although part of the coenzyme A pathway, vanin 1 (also known as pantetheinase) sits on the cell surface of many cell types as an ectoenzyme, catalyzing the breakdown of pantetheine to pantothenic acid (vitamin B5) and cysteamine, a strong reducing agent. Vanin 1 was initially discovered as a protein involved in the homing of leukocytes to the thymus. Numerous studies have shown that vanin 1 is involved in inflammation, and more recent studies have shown a key role in metabolic disease. Here, the X-ray crystal structure of human vanin 1 at 2.25 Å resolution is presented, which is the first reported structure from the vanin family, as well as a crystal structure of vanin 1 bound to a specific inhibitor. These structures illuminate how vanin 1 can mediate its biological roles by way of both enzymatic activity and protein-protein interactions. Furthermore, it sheds light on how the enzymatic activity is regulated by a novel allosteric mechanism at a domain interface. [ABSTRACT FROM AUTHOR]

Small angle X-ray scattering (SAXS) of biomacromolecules in solution has become a prominent technique in structural biology. Whilst the majority of current use is for static measurements, the field is also advancing for measurements where the sample at the beam position changes with time, using high throughput systems, chromatography, high speed mixing and pump-probe techniques in particular. Time resolved work is greatly aided by increasingly sophisticated software for acquiring and analysing data, together with developments in X-ray sources, beamline optics and detectors. The exploitation of spatial coherence is under development, with X-ray free electron lasers aiming to provide major advances in single molecule structure reconstruction and time resolution. Here we provide an overview of current developments advancing time resolved solution SAXS. [ABSTRACT FROM AUTHOR]

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction. [ABSTRACT FROM AUTHOR]

The SAXS/WAXS beamline at the Australian Synchrotron is an advanced and flexible undulator X-ray scattering beamline used for small- and wide-angle X-ray scattering analysis on a wide variety of solids, fluids and surfaces across a diverse range of research and development fields. The beamline has numerous features that minimize the intensity of the instrument background, provide automated stable optics, and allow accurate analysis of very weakly scattering samples. The geometric and intensity requirements of a three-slit collimation system are described in detail for conventional metal and single-crystal germanium slits. Straightforward ray tracing and simple linear projections describe the observed direct beam as well as parasitic background scattering geometry of the beamline at its longest camera length, providing a methodology for the design and operation of similar beamlines. As an aid to instrument design, the limit of background intensity determined by the intensity incident on single-crystal germanium guard slit edges and its q dependence was quantified at 11 keV. Details of the beamline's implementation, underlying optical concept and measured performance are given. [ABSTRACT FROM AUTHOR]

Summary: Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change. [ABSTRACT FROM AUTHOR]

Following the discovery of an exo-1,3/1,4-β-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue β- d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards β-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a β-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. Database Structural data are available in the Protein Data Bank under the accession numbers , , and [ABSTRACT FROM AUTHOR]