I20-EDE-Energy Dispersive EXAFS (EDE)
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Diamond Proposal Number(s):
[30680]
Open Access
Abstract: The increasing demand for environmentally friendly and sustainable approaches to materials synthesis calls, inter alia, for the development of biogenic methods to produce inorganic compounds by exploiting biological macromolecules and organisms. This study focuses on the optimization of a one-pot green synthesis of zinc oxide (ZnO) particles using microalgae extracts as a biogenic agent. Microalgae serve as an environmentally friendly platform for biotechnological applications due to their ability to promote the synthesis of valuable chemicals, thanks to their active components, such as enzymes. A systematic investigation of the experimental parameters revealed that both the reaction temperature and the concentration of microalgae extract significantly influenced the crystallite size of ZnO nanoparticles. In addition, the role of sodium hydroxide as a precipitating agent when used in combination with microalgae extract was addressed and compared with existing literature. The results indicate that microalgae extract can act as a scaffold to promote the controlled growth of ZnO particles. Antimicrobial tests also showed that ZnO particles synthesized with microalgae exhibited comparable antimicrobial activity with respect to ZnO produced by conventional methods. These results highlight the potential of microalgae as biogenic agents for the green synthesis of ZnO particles with tunable structural and antimicrobial properties.
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Jul 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[23942]
Open Access
Abstract: Performance plastics, such as poly(methyl methacrylate), underpin the modern economy. Global manufacturing of plastic precursors relies on fossil carbon sources, and the urgently needed shift toward renewable carbon use through biofermentation is hindered by the low tolerance of producer strains to methacrylate esters. The principal mode of butyl methacrylate cellular toxicity is membrane disruption. To understand this process, the conditions for membrane stability, and recovery after solvent shock, we investigate the phase stability of hydrated lipid membranes at high levels of a key intermediate, butyl methacrylate. We assess the role of cis- vs trans-unsaturation in 18-carbon chain phospholipids on butyl methacrylate-induced phase conversion and polymorphism. Using ssNMR, SAXS and cryoEM, we demonstrate the formation of stable lipidic cubic phases in hydrated lipid/solvent (cis-chain phospholipid lipid/butyl methacrylate) systems at a 1:6 molar ratio entirely lacking monoolein. Transient lipidic cubic phases form in trans-chain phospholipid/butyl methacrylate systems, which slowly convert to bilayers through a spontaneous “membrane healing” process during recovery after solvent shock. The observed bicontinuous nanostructures with a cubic phase architecture coexist with a stable, monocontinuous hydrated phase of the same morphology but with simpler topological connectivity, which demonstrates that phase stability in cubic phases does not require topological complementarity. We propose trans-lipid substitutions in membranes of fermentative strains as a key step toward sustainable production of methacrylate esters.
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Jul 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946, 19281]
Open Access
Abstract: Macrocyclization and multiple backbone N-methylations can significantly improve the pharmacological properties of peptides. Since chemical synthesis of such compounds is often challenging, enzyme-based production platforms are an interesting option. Here, we characterized OphP, a serine peptidase involved in the cyclization of omphalotins, a group of ribosomally produced dodecapeptides with multiple backbone N-methylations. OphP displays robust peptidase and macrocyclase activity towards multiply α-N-methylated peptides of various lengths and composition derived from the omphalotin precursor protein OphMA. In addition, OphP processes, with lower efficiency, peptides unrelated to OphMA, containing a MeGly, MeAla or Pro residue at the P1 site. Structural analysis reveals that OphP adopts a canonical prolyl oligopeptidase fold but, unlike other enzymes of this enzyme family, recognizes its substrates by their hydrophobic and multiply backbone N-methylated core rather than by the follower peptide. The activity of OphP could be harnessed for the enzymatic production of therapeutic peptides.
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Jul 2025
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B23-Circular Dichroism
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Diamond Proposal Number(s):
[33225]
Open Access
Abstract: Deep eutectic solvents (DESs) have emerged as powerful environments to enhance enzymatic reactions, formulate therapeutic proteins, and develop protein-based biomaterials. Despite the wide range of properties that could be achievable through the compositional design of DESs, protein solubilization only happens in a relatively narrow range of hydrophilic DESs. Here, we use surface-modification for the generalized solubilization of proteins in both hydrophilic and hydrophobic DESs. Using surface-modified myoglobin as a model, we show that both DES polarity and hydrogen bond capacity play important roles in dictating the conformational state of the protein. In the hydrophilic DES the protein displays a near-native conformation with an improvement of the thermal stability of + 28 °C compared to aqueous solutions. In contrast, hydrophobic DESs stabilize partially folded intermediates which can refold from temperatures as high as 190 °C. As such, our approach provides a platform to generalize protein incorporation into anhydrous DESs that could be exploited in biocatalysis, biomolecule stabilization, and biomaterials.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32544]
Open Access
Abstract: The recent discovery of the PETase enzyme family offers a sustainable solution for depolymerizing poly(ethylene terephthalate) (PET), one of the most widespread plastic compounds, under mild conditions. This enables the environmentally beneficial conversion of plastic waste into value-added products. Among this enzyme family, PETase from Ideonella sakaiensis has been the most extensively studied. Although other similar enzymes have been discovered, our knowledge about the catalytic and structural properties of this class remains limited. In this study, a PETase-like enzyme (PETase SM14) from Streptomyces sp. SM14 was heterologously produced in Escherichia coli, and its activity was tested on post-consumer plastic substrates using high-performance liquid chromatography for product quantification as well as scanning electron microscopy and atomic force microscopy for substrate surface imaging evaluation. PETase SM14 exhibited high salt tolerance (1.5 M), good heat resistance (Tm 56.26 °C), and optimal activity at pH 9.0, highlighting its potential for PET waste bioremediation. Furthermore, its X-ray crystal structure was solved at 1.43 Å resolution, revealing conserved features of the PETase family with potential relevance for future engineering applications.
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May 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: The prodigious ability of bacteria to catabolize aromatic compounds has sparked considerable efforts to engineer bacteria to valorize lignin, an under-utilized component of biomass. Despite decades of study, key catabolic pathways and enzymes remain poorly characterized. We recently identified the hydroxyphenylethanone (Hpe) pathway, which enables Rhodococcus rhodochrous GD02 and other bacteria to catabolize 4-hydroxyacetophenone (HAP) and acetovanillone (AV), which are generated in the catalytic fractionation of lignin. Catabolism is initiated by a two-component, ATP-dependent dikinase, HpeHI, homologs of which are involved in the catabolism of other aromatic compounds. In biochemical studies, the kinase activity of HpeHI was highest at low ionic strength and low concentrations of Mn2+. HpeHI had highest apparent specificity for HAP and AV (kcat/KM ≥ 250 mM-1 s-1) and had submicromolar KM values for these substrates, consistent with the enzyme acting as a scavenging system. The enzyme also transformed 4-hydroxybenzaldehyde, vanillin, acetosyringone, and phenol. A 1.8 Å crystal structure of HpeI revealed that it is homologous to the ATP-grasp domain of rifampin phosphotransferase (RPH) while an AlphaFold model of HpeH indicated that it is homologous to the swivel and rifampin-binding domains of RPH. Consistent with HpeHI using a similar mechanism where the swivel domain transits between the spatially distinct substrate-binding sites, substitution of the conserved His residue in HpeH abolished kinase activity. Moreover, the HpeH component alone catalyzed phosphotransfer from 4-phosphoacetophenone to AV. This study reveals a subfamily of small molecule dikinases that comprise two components, some of which are involved in aromatic compound catabolism.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[32736]
Open Access
Abstract: Enantioselective reduction of hydrazones provides a convergent and versatile route to synthesize hydrazine-containing motifs that are commonly found in pharmaceuticals and agrochemicals. However current methods require the use of precious metals, costly chiral ligands and/or forcing reaction conditions. Here, we report the development of a biocatalytic approach for enantioselective hydrazone reduction using engineered imine reductases. Following evaluation of an in-house panel of >400 IRED sequences, we identified a single IR361 I127F L179V variant that promotes reduction of Cbz-protected hydrazones. Introduction of an additional two mutations via directed evolution afforded HRED1.1 that is 20-fold more active than the parent template and promotes reduction of a variety of protected hydrazones in high yields and selectivities (>99% e.e.), including in preparative scale biotransformations. Structural analysis of HRED1.1 provides insights into the origins of its unique hydrazone reductase activity. This study offers a powerful biocatalytic route to synthesize valuable chiral hydrazine products and further expands the impressive range of transformations accessible with engineered imine reductases.
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Apr 2025
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B21-High Throughput SAXS
I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[31800]
Open Access
Abstract: Stimuli-responsive polymeric vesicles offer a versatile platform for mimicking dynamic cell-like behaviors for synthetic cell applications. In this study, thermally responsive polymeric droplets derived from poly(ethylene oxide)-poly(butylene oxide) (PEO-PBO) polymersomes, aiming to create synthetic cell models that mimic key biological functions are developed. Upon heating, the nanoscale vesicles undergo fusion, transforming into sponge-like microscale droplets enriched with membrane features. By modulating the temperature, these droplets display dynamic properties such as contractility, temperature-induced fusion, and cargo trapping, including small molecules and bacteria, thereby demonstrating their ability to dynamically interface with biological entities. The findings demonstrate the potential of our sponge-like droplets in synthetic cell applications, contributing to the understanding of PEO-PBO polymersomes’ unique characteristics, expanding the capabilities of synthetic cell structures, and representing an exciting possibility for advancing soft matter engineering to cell-like behaviors.
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Mar 2025
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I03-Macromolecular Crystallography
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Andry Mercedes
Mavila
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Jhon Antoni
Vargas
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Eloy
Condori
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Erick Giancarlo
Suclupe Farro
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Adriano
Alves Furtado
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Josué Manuel
López
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Silvia Lucila
Gonzalez
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Humberto D’muniz
Pereira
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Jorge Luis
Marapara
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Roger Ruiz
Paredes
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Marianela
Cobos
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Juan C.
Castro
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Richard Charles
Garratt
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Diego Antonio
Leonardo
Diamond Proposal Number(s):
[31229]
Abstract: Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg2+-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
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Mar 2025
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Krios I-Titan Krios I at Diamond
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Lee
Sherry
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Keith
Grehan
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Mohammad W.
Bahar
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Jessica J.
Swanson
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Helen
Fox
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Sue
Matthews
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Sarah
Carlyle
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Ling
Qin
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Claudine
Porta
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Steven
Wilkinson
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Suzanne
Robb
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Naomi
Clark
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John
Liddell
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Elizabeth E.
Fry
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David I.
Stuart
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Andrew J.
Macadam
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David J.
Rowlands
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Nicola J.
Stonehouse
Diamond Proposal Number(s):
[28713]
Open Access
Abstract: The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses concerns, due to the potential for virus escape during vaccine manufacture. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV2 mutant, termed SC5a, in comparison to PV2-SC6b VLPs described previously and investigated the immunogenicity of PV2-SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.
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Mar 2025
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