B21-High Throughput SAXS
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Diamond Proposal Number(s):
[29895]
Open Access
Abstract: A lipopeptide is designed that contains an epitope from simian virus T-antigen (SV40T, PKKKRKV) conjugated to an N-terminal palmitoyl (C16-) moiety, with the aim to act as an effective cell-penetrating lipopeptide, with additional aggregation propensity conferred by the lipid chain. A combination of cryo-TEM and small-angle X-ray scattering (SAXS) is used to show that the lipopeptide forms micelles, but mixtures with DNA lead to formation of fractal cluster-like co-assemblies due to intercalation of the DNA and peptide. Spectroscopic studies using fluorescence and circular dichroism (along with fiber X-ray diffraction) show that the peptide interacts with DNA and inserts into the groove. Confocal microscopy along with flow cytometry confirms delivery of DNA into both HeLa and mouse embryonic stem cells (mESCs) in pluripotent state, and the system shows excellent cytocompatibility as confirmed by MTT assays. Our data indicate that the lipopeptide may outperform the DNA transfection agent lipofectamine in DNA delivery into these stem cells and it enables DNA delivery into the cytoplasm and nucleus.
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Mar 2026
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VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[28534, 34263]
Open Access
Abstract: Schistosoma mansoni cathepsin D1 (SmCD1) has been shown to be an essential enzyme for helminth metabolism due to its role in haemoglobin degradation: a key amino-acid source for the developing parasite. Therefore, the enzyme is a potential target for the development of antischistosomal inhibitors. SmCD1 has significant sequence identity to cathepsin D-like proteases found in other schistosome species and homology to mammalian aspartic proteases. Here, we report the first crystal structures of a helminth cathepsin D, SmCD1, and have identified a single-domain antibody (nanobody) that specifically binds to SmCD1 with nanomolar affinity but does not recognize human cathepsin D. We have mapped the epitope of the nanobody by determining the crystal structure of the enzyme–nanobody complex, revealing the conformation of SmCD1 in the propeptide-bound state.
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Feb 2026
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I04-Macromolecular Crystallography
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Qiangqiang
Wei
,
Ashley J.
Taylor
,
Nagaraju
Miriyala
,
Mahesh A.
Barmade
,
Zachary O.
Gentry
,
Jordan
Anderson-Daniels
,
Kevin B.
Teuscher
,
Mackenzie M.
Crow
,
Chideraa
Apakama
,
Taylor M.
South
,
Tyson A.
Rietz
,
Kangsa
Amporndanai
,
Jason
Phan
,
John L.
Sensintaffar
,
Mark
Denison
,
Taekyu
Lee
,
Stephen W.
Fesik
Open Access
Abstract: The papain-like protease (PLPro) plays a key role in SARS-CoV-2 replication and represents a promising target for the development of new antiviral therapies. Previous efforts to develop fragment-derived inhibitors of PLPro led to the identification of a novel class of spiro[chromane-2,4′-piperidin]-4-one inhibitors exemplified by lead compound 7. High-resolution covalent cocrystal structures and molecular dynamics simulations were utilized to guide the development of a series of low-nanomolar irreversible PLPro inhibitors, with lead compound 45 demonstrating strong enzymatic inhibition (IC50 = 0.059 μM at T = 60 min) and antiviral activity in A549 cells (EC50 = 2.1 μM at 48 hpi). This novel class of inhibitors represents a promising avenue for the development of therapeutics to overcome the potential of drug-resistant viral strains and future coronavirus outbreaks.
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Feb 2026
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440, 37593]
Open Access
Abstract: Angiotensin I-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase involved in blood pressure regulation through proteolysis of angiotensin I (Ang-I) into the potent vasoconstrictor, angiotensin II (Ang-II). Inhibition of ACE is therefore used for the treatment of hypertension, heart failure, myocardial infarction, stroke and chronic kidney disease. Current ACE inhibitors (ACEi) bind both the N- and C-catalytic domains of ACE (referred to as nACE and cACE), and this has been linked to the occurrence of side effects due to the wide substrate specificity of ACE. The development of domain selective ACEi with reduced side effects is therefore key for improved therapeutic intervention. Understanding how current ACEi bind nACE and cACE, and their differences in domain selectivity should aid structure-based development of more selective ACEi by identifying different chemical groups that increase or decrease selectivity. We present the kinetic and structural characterisation of nACE and cACE with three thiolate ACEi, captopril (Ki, nACE = 2.53 nm and cACE = 2.04 nm), rentiapril (monomer Ki, nACE = 2.22 nm and cACE = 6.77 nm) and zofenoprilat (Ki, nACE = 2.86 nm and cACE = 0.61 nm). Detailed structural analysis indicated that the S2′ subsite likely contributes to the variation in domain selectivity observed for rentiapril and zofenoprilat due to differences in hydrophobicity and displacement of water molecules that contribute to ACE's hydration shell. Interestingly, in the cACE crystal structure, rentiapril bound as a dimer, and kinetic data revealed that both the monomeric and dimeric (dimer Ki, nACE = 15.11 nm and cACE = 36.38 nm) forms of rentiapril inhibit ACE with nanomolar affinity.
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Feb 2026
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I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[31906, 39961]
Open Access
Abstract: Mercury (Hg) is a global environmental concern due to its microbial conversion to methylmercury (MeHg), a potent neurotoxin that bioaccumulates in food webs and poses risks to ecosystems and human health. Thiol functional groups (RSH) play an important role in controlling Hg(II) speciation and bio-uptake in methylating bacteria, yet the spatial distribution and density of these thiols within cells remain largely unknown. We isolated subcellular fractions of the Hg methylating bacterium Geobacter sulfurreducens in the exponential growth phase, and used Hg LIII-edge EXAFS (Extended X-ray Absorption Fine Structure) to quantify thiols in the extracellular medium, inner and outer membranes, periplasm and cytoplasm. The whole-cell thiol content was determined to be 1.3 × 10−10 μmol cell−1. The inner membrane contributed 7.1 × 10−11 (53%), the outer membrane 1.2 × 10−11 (9%), the periplasm 3.6 × 10−11 (27%) and the cytoplasm 1.5 × 10−11 μmol cell−1 (11%). The extracellular fraction contributed an additional 5.7 × 10−11 μmol cell−1, corresponding to 30% of the thiols of the cell culture. Local thiol density (thiols normalized to TOC in individual compartment, RSH/TOC, μmol g−1 C) was 36, 450, 140, 600 and 29 μmol g−1 C in the cytoplasm, inner membrane, periplasm, outer membrane and extracellular fractions, respectively. EXAFS analyses demonstrate Hg-thiolate coordination across all compartments, with Hg-O/N bonding and elemental Hg0 formed at higher Hg loadings. In the periplasm, Hg-disulfide and traces of β-HgS were detected. The high thiol density at the membranes, relative to other compartments, may imply they have an important role in the retention and internalization of Hg(II). Periplasmic thiols may modulate Hg(II) transfer between membranes, and cytoplasmic thiols may regulate the intracellular availability of Hg(II) for methylation. This work provides the first compartment-resolved quantification of thiol abundances and densities in a model Hg-methylating bacterium at subcellular level, offering a mechanistic framework for understanding the speciation, bioavailability, and subcellular transformation of Hg(II) with relevance for other soft metals (e.g., Cd, Pb, Zn, Ag, and Cu).
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Feb 2026
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[38262]
Open Access
Abstract: Pseudomonas putida is a plant-beneficial rhizobacterium that encodes multiple type-VI secretion systems (T6SS) to outcompete phytopathogens in the rhizosphere. Among its antibacterial effectors, Tke5 (a member of the BTH_I2691 protein family) is a potent pore-forming toxin that disrupts ion homeostasis without causing considerable membrane damage. Tke5 harbours an N-terminal MIX domain, which is required for T6SS-dependent secretion in other systems. Many MIX domain-containing effectors require T6SS adaptor proteins (Tap) for secretion, but their molecular mechanisms of adaptor-effector binding remain elusive. Here, we report the 2.8 Å cryo-EM structure of the Tap3-Tke5 complex of P. putida strain KT2440, providing structural and functional insights into how effector Tke5 is recruited by its cognate adaptor protein Tap3. Functional dissection shows that the α-helical region of Tke5 is sufficient to kill intoxicated bacteria, while its β-rich region likely contributes to target membrane specificity. These findings delineate a mechanism of BTH_I2691 proteins for Tap recruitment and toxin activity, contributing to our understanding of a widespread yet understudied toxin family.
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Jan 2026
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[34223]
Open Access
Abstract: DNA polymerase β (Pol β) is an important polymerase that functions in DNA repair within the Base Excision Repair and Non-Homologous End-Joining pathways. It is estimated to function in the repair of up to 50,000 DNA lesions per cell per day, within the base excision repair pathway (BER). Given the significant role Pol β plays in repairing DNA, genetic variants of Pol β have the potential to perturb repair, resulting in mutation accumulation which can potentiate cancer formation. Here we identify an unstudied human germline variant of Pol β, the S180R variant (rs1585898410), which introduces a significant amino acid alteration within the dNTP binding pocket of the enzyme. We demonstrate that S180R is a low fidelity variant of Pol β due to its loss of the ability to discriminate correct nucleotides from incorrect nucleotides. We also show that this variant exhibits a much slower rate of nucleotide incorporation, which could further disrupt repair capacity in vivo. Structural data reveal that this variant not only has structural changes that may disrupt dNTP binding but also a loss of primer terminus positioning and dynamic flexibility of the fingers domain in the binary state, which likely are driving the low fidelity of S180R Pol β. This study highlights the importance of binary positioning and nucleotide coordinating residues for maintaining nucleotide selectivity, polymerase function, and fidelity. It also emphasizes the importance of further study of this human germline Pol β variant in vivo.
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Jan 2026
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B21-High Throughput SAXS
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María Florencia
Pignataro
,
Natalia Brenda
Fernández
,
Alba
Garay-Alvarez
,
María Florencia
Pavan
,
Rafael
Molina
,
Ines G.
Munoz
,
Julián
Grossi
,
Martín
Noguera
,
Antonella
Vila
,
Augusto E.
García
,
Hernán G.
Gentili
,
Naira Antonia
Rodríguez
,
Martín
Aran
,
Viviana
Parreño
,
Marina
Bok
,
Juan A.
Hermoso
,
Lorena Itatí
Ibañez
,
Javier
Santos
Diamond Proposal Number(s):
[35926]
Open Access
Abstract: Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich’s ataxia (FRDA).
In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway.
As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.
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Jan 2026
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I24-Microfocus Macromolecular Crystallography
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Lídia Dos Passos
Lima
,
Dev
Sriranganadane
,
Daiane Laise
Da Silva
,
Natália C.
Drebes Dörr
,
Enzo Breviglieri Sichi
Mello
,
Caio Vinicius
Dos Reis
,
Rogério Ferreira
Lourenço
,
José Felipe Teixeira Da Silva
Santos
,
Anita
Salmazo
,
Brenno Wendler
Miranda
,
Katlin B.
Massirer
,
Rafael M.
Counago
,
Cristina E.
Alvarez-Martinez
Open Access
Abstract: Serine/threonine (Ser/Thr) kinases of the Hanks-type family are widespread in bacteria, playing key roles in signal transduction. The transmembrane Ser/Thr kinase PknS (XAC4127) from the phytopathogenic bacterium Xanthomonas citri is required for the expression of a type VI secretion system, which confers resistance to predation by the soil amoeba Dictyostelium discoideum. PknS exerts its function via activation of the cognate ECF-type alternative sigma factor EcfK, ultimately triggering the expression of type VI secretion system (T6SS) genes. In this study, we characterize PknS, demonstrating its ability to undergo autophosphorylation both in vitro and within X. citri cells. Structural analysis of the PknS kinase domain revealed the conservation of the canonical fold characteristic of Hanks-type kinases. PknS directly phosphorylates EcfK at five Ser/Thr residues located in two distinct regions of the sigma factor: the conserved σ2 domain (residue T51) and a nonconserved linker connecting domains σ2 and σ4 (residues T104, T106, S108, and S110). The conserved residue T51, previously shown to be essential for sigma factor activity in an EcfK homolog, corresponds to a site that directly interacts with the RNA polymerase β′ subunit. Site-directed mutagenesis analyses further revealed that the conserved residue T106 is also critical for EcfK function. Structural studies indicated that, in addition to T51, phosphorylation at T106 activates EcfK by promoting its interaction with a positively charged pocket within the RNA polymerase β′ subunit. Collectively, our findings describe a previously unknown signal transduction pathway involving a Hanks-type kinase and a sigma factor, providing new insights into the mechanisms of sigma factor activation via phosphorylation in bacteria.
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Jan 2026
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I03-Macromolecular Crystallography
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Open Access
Abstract: Aromatic polyketides from Actinobacteria are structurally complex bioactive natural products with significant therapeutic potential, whose biosynthesis involves polyketide chain assembly, keto reduction, cyclization, and aromatization. This is followed by pathway-specific enzymatic tailoring steps, occasionally including rare oxidative rearrangements of the carbon skeleton, as exemplified by the rishirilides. In this study, we investigate RslO9, a flavin-dependent tailoring key enzyme of rishirilide biosynthesis, previously hypothesized to facilitate a lactone-forming Baeyer–Villiger oxidation of the rishirilide naphthoquinone core and subsequent intramolecular aldol condensation. Through detailed investigation of RslO9’s mechanism, structural features, and substrate scope, we unexpectedly found that the naphthoquinone moiety of the non-natural substrate lapachol undergoes hydroxylation followed by a benzilic acid rearrangement, producing the Hooker intermediate–a hallmark of the intricate Hooker oxidation. Our data support a similar alkyl migration mechanism for RslO9’s native substrate, upending its prior classification as a Baeyer–Villiger monooxygenase and challenging the proposed role of related enzymes while also providing a novel framework for exploring their catalytic roles.
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Jan 2026
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