I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[22637]
Open Access
Abstract: MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.
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May 2022
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E01-JEM ARM 200CF
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Diamond Proposal Number(s):
[26559]
Abstract: We present a trainable segmentation method implemented within the python package ParticleSpy. The method takes user labelled pixels, which are used to train a classifier and segment images of inorganic nanoparticles from transmission electron microscope images. This implementation is based on the trainable Waikato Environment for Knowledge Analysis (WEKA) segmentation, but is written in python, allowing a large degree of flexibility and meaning it can be easily expanded using other python packages. We find that trainable segmentation offers better accuracy than global or local thresholding methods and requires as few as 100 user-labelled pixels to produce an accurate segmentation. Trainable segmentation presents a balance of accuracy and training time between global/local thresholding and neural networks, when used on transmission electron microscope images of nanoparticles. We also quantitatively investigate the effectiveness of the components of trainable segmentation, its filter kernels and classifiers, in order to demonstrate the use cases for the different filter kernels in ParticleSpy and the most accurate classifiers for different data types. A set of filter kernels is identified that are effective in distinguishing particles from background but that retain dissimilar features. In terms of classifiers, we find that different classifiers perform optimally for different image contrast; specifically, a Random Forest classifier performs best for high-contrast ADF images, but that QDA and Gaussian Naïve Bayes classifiers perform better for low-contrast TEM images.
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May 2022
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I24-Microfocus Macromolecular Crystallography
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Chih-Jen
Wei
,
Wei
Bu
,
Laura A.
Nguyen
,
Joseph D.
Batchelor
,
Junghyun
Kim
,
Stefania
Pittaluga
,
James R.
Fuller
,
Hanh
Nguyen
,
Te-Hui
Chou
,
Jeffrey I.
Cohen
,
Gary J.
Nabel
Diamond Proposal Number(s):
[22894]
Abstract: Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[27031]
Abstract: The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 binds angiotensin converting enzyme-2 (ACE-2) on the surface of epithelial cells, leading to fusion, and entry of the virus into the cell. This interaction can be blocked by the binding of llama-derived nanobodies (VHHs) to the RBD, leading to virus neutralisation. Structural analysis of VHH-RBD complexes by X-ray crystallography enables VHH epitopes to be precisely mapped, and the effect of variant mutations to be interpreted and predicted. Key to this is a protocol for the reproducible production and crystallization of the VHH-RBD complexes. Based on our experience, we describe a workflow for expressing and purifying the proteins, and the screening conditions for generating diffraction quality crystals of VHH-RBD complexes. Production and crystallization of protein complexes takes approximately twelve days, from construction of vectors to harvesting and freezing crystals for data collection.
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May 2022
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10369]
Abstract: The Streptococcus pneumoniae Rgg144/SHP144 regulator-peptide quorum sensing (QS) system is critical for nutrient utilisation, oxidative stress response, and virulence. Here we characterised this system by assessing the importance of each residue within the active short hydrophobic peptide (SHP) by alanine-scanning mutagenesis and testing the resulting peptides for receptor binding and activation of the receptor. Interestingly, several of the mutations had little effect on binding to Rgg144 but reduced transcriptional activation appreciably. In particular, a proline substitution (P21A) reduced transcriptional activation by 29-fold but bound with 3-fold higher affinity than the wild-type SHP. Consistent with the function of Rgg144, the mutant peptide led to decreased utilisation of mannose and increased susceptibility to superoxide generator paraquat. Pangenome comparison showed full conservation of P21 across SHP144 allelic variants. Crystalization of Rgg144 in the absence of peptide revealed a comparable structure to the DNA bound and free forms of its homologues suggesting similar mechanisms of activation. Together, these analyses identify key interactions in a critical pneumococcal QS system. Further manipulation of the SHP has the potential to facilitate the development of inhibitors that are functional across strains. The approach described here is likely to be effective across QS systems in multiple species.
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May 2022
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E02-JEM ARM 300CF
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Diamond Proposal Number(s):
[22479]
Open Access
Abstract: Li-rich metal oxides, such as Li1.2Ni0.13Mn0.54Co0.13O2, can deliver high specific capacities because of the redox of lattice O2− in addition to the cations. Observing oxygen distortions is key to understand the redox process. Electron ptychography is a phase-reconstruction method in 4D scanning transmission electron microscopy, providing atomic-resolution phase images with high signal-to-noise ratio and dose efficiency. Herein, we use electron ptychography to image the oxygen shift in Li1.2Ni0.13Mn0.54Co0.13O2 during the first cycle. The picometer-scale precision measurement shows distinct oxygen shifts in the bulk and surface after charging and compares with various theoretical anionic redox models. The shift after discharging is not seen to recover in the bulk accounting for voltage hysteresis; however, it recovers close to the surface, although with a phase change. We suggest that Li1.2Ni0.13Mn0.54Co0.13O2 proceeds distinct oxygen redox in the bulk and surface. The altered oxygen sublattice after first cycle potentially explains the changed voltage profiles of following cycles.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19458, 23459]
Open Access
Abstract: β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.
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May 2022
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I19-Small Molecule Single Crystal Diffraction
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Diamond Proposal Number(s):
[23480]
Open Access
Abstract: Herein, we report on the use of tetrathiavulvalene-tetrabenzoic acid, H4TTFTB, to engender semiconductivity in porous hydrogen-bonded organic frameworks (HOFs). By tuning the synthetic conditions, three different polymorphs have been obtained, denoted MUV-20a, MUV-20b, and MUV-21, all of them presenting open structures (22, 15, and 27%, respectively) and suitable TTF stacking for efficient orbital overlap. Whereas MUV-21 collapses during the activation process, MUV-20a and MUV-20b offer high stability evacuation, with a CO2 sorption capacity of 1.91 and 1.71 mmol g–1, respectively, at 10 °C and 6 bar. Interestingly, both MUV-20a and MUV-20b present a zwitterionic character with a positively charged TTF core and a negatively charged carboxylate group. First-principles calculations predict the emergence of remarkable charge transport by means of a through-space hopping mechanism fostered by an efficient TTF π–π stacking and the spontaneous formation of persistent charge carriers in the form of radical TTF•+ units. Transport measurements confirm the efficient charge transport in zwitterionic MUV-20a and MUV-20b with no need for postsynthetic treatment (e.g., electrochemical oxidation or doping), demonstrating the semiconductor nature of these HOFs with record experimental conductivities of 6.07 × 10–7 (MUV-20a) and 1.35 × 10–6 S cm–1 (MUV-20b).
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May 2022
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I04-Macromolecular Crystallography
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Open Access
Abstract: The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in D. dadantii, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sialic acids terminate many N- and O-glycans and are widely distributed on cell surfaces. There are a diverse range of enzymes which interact with these sugars throughout the tree of life. They can act as receptors for influenza and specific betacoronaviruses in viral binding and their cleavage is important in virion release. Sialic acids are also exploited by both commensal and pathogenic bacteria for nutrient acquisition. A common modification of sialic acid is 9-O-acetylation, which can limit the action of sialidases. Some bacteria, including human endosymbionts, employ esterases to overcome this modification. However, few bacterial sialic acid 9-O-acetylesterases (9-O-SAEs) have been structurally characterized. Here, the crystal structure of a 9-O-SAE from Phocaeicola vulgatus (PvSAE) is reported. The structure of PvSAE was determined to resolutions of 1.44 and 2.06 Å using crystals from two different crystallization conditions. Structural characterization revealed PvSAE to be a dimer with an SGNH fold, named after the conserved sequence motif of this family, and a Ser–His–Asp catalytic triad. These structures also reveal flexibility in the most N-terminal α-helix, which provides a barrier to active-site accessibility. Biochemical assays also show that PvSAE deacetylates both mucin and the acetylated chromophore para-nitrophenyl acetate. This structural and biochemical characterization of PvSAE furthers the understanding of 9-O-SAEs and may aid in the discovery of small molecules targeting this class of enzyme.
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May 2022
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