I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: hiamine-dependent actinobacterial 2-hydroxyacyl-CoA lyase (AcHACL) catalyzes the reversible cleavage of 2-hydroxyacyl-CoAs to formyl-CoA and carbonyl compounds. To exploit the enzyme's biotechnological potential, a deeper understanding of the catalysis is required. Previously, AcHACL E493 was identified as an important acid/base catalyst. Here, wild-type and E493 mutant crystal structures representing Michaelis complexes with 2-hydroxyisobutyryl-CoA and (S)-2-methylglyceryl-CoA are provided. Although E493 guarantees high rates of essential proton transfers in AcAHCL-catalyzed on-pathway cleavage of 2-hydroxyacyl-CoAs and off-pathway carboligations with short-chain aldehydes and ketones, wild-type substrate accommodation is suboptimal. Not E493D, but E493A and E493S mutations improved KM. However, kcat is substantially reduced in the mutants. These tradeoffs are discussed by comparing active sites of AcHACL and related enzymes either lacking or possessing an E493 homolog.
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Jan 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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I03-Macromolecular Crystallography
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Qinglong
Meng
,
Caecilie
Benckendorff
,
Charlotte
Morrill
,
Ying
Zhuo
,
Annette
Egerström
,
Aisling
Ní Cheallaigh
,
Sasha R.
Derrington
,
Richard
Obexer
,
Mary
Ortmayer
,
Colin W.
Levy
,
James D.
Finnigan
,
Simon J.
Charnock
,
Nicholas J.
Turner
,
Gavin J.
Miller
,
Sarah L.
Lovelock
Diamond Proposal Number(s):
[31850]
Open Access
Abstract: The rapid emergence of RNA therapeutics has highlighted the need for more efficient, scalable and sustainable methods for their manufacture. Biocatalytic approaches hold particular promise, but rely on a secure, sustainable and low-cost supply of nucleoside triphosphate (NTP) building blocks, including those containing chemical modifications. Here we report the development of a biocatalytic approach and engineered enzymes to convert widely available nucleosides into NTPs featuring pharmaceutically relevant modifications using inexpensive phosphate donors. Importantly our strategy obviates the need for ATP as a phosphate donor that complicates NTP isolation using existing methods. To showcase the utility of our approach, we employ an engineered acid phosphatase, polyphosphate kinase and acetate kinase to produce 2′-O-methoxyethyl-ATP (2′-MOE-ATP) and 2′-fluoro-ATP, key building blocks of commercial therapeutics. Finally, we show that crude NTPs from our process can be used directly in enzymatic oligonucleotide synthesis, obviating the need for costly NTP isolation or purification steps.
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Dec 2025
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[31371]
Open Access
Abstract: Magnetotactic bacteria, such as Magnetospirillum gryphiswaldense MSR-1, naturally produce magnetosomes—intracellular magnetic nanoparticles that enable navigation within geomagnetic fields. Magnetosomes hold significant potential for biomedical and biotechnological applications; however, key aspects of their biomineralization remain poorly understood. This study investigates how oxidative stress, induced by hydrogen peroxide and iron, influences magnetosome formation and bacterial physiology under aerobic and microaerobic conditions. Single-cell advanced microscopy and high-throughput techniques revealed that microaerobic conditions supported robust magnetosome production and larger magnetite crystals while maintaining low oxidative stress levels. In contrast, aerobic conditions suppressed magnetosome formation, reduced intracellular iron content, and increased reactive oxygen species (ROS) levels. High extracellular iron enhanced the formation of longer magnetosome chains in microaerobic cultures without causing toxicity but reduced cell viability under aerobic conditions. Hydrogen peroxide exposure caused mild damage and a 25% viability drop in magnetosome-producing cells but led to severe damage and an 80% viability drop in non-magnetosome-producing cells, along with chain fragmentation and smaller magnetite crystals. These results suggest that magnetosome-producing cells exhibit greater resilience to oxidative stress, potentially due to ROS scavenging properties of magnetosomes, and highlight the intricate interplay between oxidative stress, iron regulation, and magnetosome biomineralization. Single-cell analysis revealed heterogeneity in physiological responses, further demonstrating the complexity of these processes. These findings underscore the importance of monitoring physiological changes during production processes to enhance the efficiency and robustness of magnetosome synthesis. The insights gained provide a foundation for improving bioprocesses for large-scale production of high-quality magnetosomes, advancing their applications in biomedicine and biotechnology.
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Dec 2025
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Hui
Sun
,
Yanan
Jiang
,
Miaolin
Lan
,
Ming
Zhou
,
Gangshun
Yi
,
Juan
Shen
,
Tingting
Deng
,
Liqin
Liu
,
Yang
Huang
,
Yu
Li
,
Jinfu
Su
,
Yanling
Lin
,
Zhenqin
Chen
,
Lizhi
Zhou
,
Tingting
Li
,
Hai
Yu
,
Tong
Cheng
,
Yali
Zhang
,
Lunzhi
Yuan
,
Shaowei
Li
,
Ying
Gu
,
Peijun
Zhang
,
Ningshao
Xia
,
Qingbing
Zheng
Open Access
Abstract: The rapid evolution of SARS-CoV-2 and the subsequent emergence of Omicron subvariants pose significant challenges to the efficacy of existing vaccines and therapeutics, including those previously reported most broad neutralizing antibodies (bnAbs). Here, we investigated the molecular basis of the altered neutralization profile of a bnAb, 1C4, against recent variants. 1C4 is effective against early variants from Alpha to Omicron BQ.1, but is circumvented by BQ.1.1, XBB and thereafter variants, primarily due to an additional R346T mutation that diminishes its binding affinity. Cryo-electron microscopy analysis revealed that despite the loss of neutralizing potency, 1C4 retained residual binding to the spike protein of immune-evasive variants such as XBB, which harbor altered receptor-binding domain (RBD). Furthermore, 1C4 exhibited a diminished capacity to inhibit ACE2 engagement with Omicron variants, amplifying the intricacies of viral immune evasion tactics. To address this, we employed the mi3-SpyCatcher-based nanoparticle to polymerize 1C4 (mi3-1C4), which reestablished the neutralization potency against recent variants by enhancing avidity via multivalent binding. Such multivalent binding can promote efficient spike aggregation as well as viral cross-linking, thereby providing enhanced protection against both the infection of Beta and XBB variants in a hamster model. Together, our findings delineate the molecular landscape of immune evasion by neutralizing antibodies and provide strategic insight for the adaptation of antibody engineering to keep pace with viral evolution.
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Dec 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26779]
Open Access
Abstract: NTF2-like proteins are compact α + β fold domains with cone-shaped architectures and internal pockets, making them attractive scaffolds for the de novo design of small-molecule binders and enzymes. However, creating ligand-binding pockets often compromises folding stability, posing a key challenge in de novo protein design. Here, we introduce strategies to stabilize NTF2-like domains while preserving pocket geometry and accessibility. By expanding the hydrophobic core through computationally designed α-helical subdomains or homodimer interfaces buttressing the β-sheet's convex face, we enhance structural stability without blocking pocket access on the concave face. Biochemical, biophysical, and crystallographic analyses confirm that the designed buttressing elements maintain the intended fold and support diverse, well-formed hydrophobic ligand-binding pockets with increased preorganization. Our results demonstrate that structural stabilization and pocket optimization need not be mutually exclusive, providing a generalizable approach to create robust ligand-binding proteins. This framework addresses a major bottleneck in protein design and should fuel the development of NTF2-based scaffolds for applications in small-molecule biosensing and enzyme catalysis.
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Nov 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035, 29470]
Open Access
Abstract: Advanced cell therapies require robust matrices for enhanced efficacy and delivery, but fabricating cell-specific hydrogels with strong tissue adhesiveness remains challenging. Cell membrane engineering offers a non-genetic strategy to modify cell surfaces and improve therapeutic properties. This study reports an artificial membrane-binding protein (AMBP), [cat.mTG(S)], that drives in situ formation of proteinaceous hydrogels on the plasma membrane of human dermal fibroblasts (HDFs). The AMBP is created by chemically supercharging (cationizing) microbial transglutaminase (mTG) and then electrostatically complexing it with an anionic polymer-surfactant (S). Biophysical studies confirm that this polymer surfactant complexation stabilizes the enzyme's structure and partially restores its activity lost during cationization. [cat.mTG(S)] effectively labels HDF plasma membranes with low cytotoxicity, unlike unmodified mTG (no binding) or cationized mTG (internalized). Live-cell confocal microscopy demonstrates that [cat.mTG(S)] on HDFs successfully cross-links external proteins into robust hydrogels extending beyond the cell surface and bridging cells, maintaining high cell viability. This AMBP provides a novel, non-genetic approach for localized, cell-surface engineering, enabling direct creation of protective and interactive hydrogel microenvironments for advanced cell-based therapies.
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Nov 2025
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[28576]
Open Access
Abstract: Carboxysomes in cyanobacteria and certain proteobacteria enable efficient CO2 fixation by encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a semipermeable shell. Sequestered CA catalyze the rapid interconversion of CO2 and HCO3−, supplying elevated levels of CO2 to boost Rubisco carboxylation. Despite its essential role, the structure and encapsulation of CA within carboxysomes remain poorly understood. Here, we determined the molecular structure of α-carboxysomal CA from the model chemoautotrophic bacterium Halothiobacillus neapolitanus (HnCsoSCA). HnCsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that HnCsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. HnCsoSCA truncations suggest nonspecific interactions between HnCsoSCA and CsoS1A. We further show that HnCsoSCA bridges Rubisco and the shell facets. Our study offers insights into the assembly and encapsulation mechanisms of α-carboxysomes and provides the framework for reprogramming carboxysome structures for synthetic biology and biotechnological applications.
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Nov 2025
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I03-Macromolecular Crystallography
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Open Access
Abstract: Artificial metalloenzymes present a promising avenue for abiotic catalysis within living systems. However, their in vivo application is currently limited by critical challenges, particularly in selecting suitable protein scaffolds capable of binding abiotic cofactors and maintaining catalytic activity in complex media. Here we address these limitations by introducing an artificial metathase—an artificial metalloenzyme designed for ring-closing metathesis—for whole-cell biocatalysis. Our approach integrates a tailored metal cofactor into a hyper-stable, de novo-designed protein. By combining computational design with genetic optimization, a binding affinity (KD ≤ 0.2 μM) between the protein scaffold and cofactor is achieved through supramolecular anchoring. Directed evolution of the artificial metathase yielded variants exhibiting excellent catalytic performance (turnover number ≥1,000) and biocompatibility. This work represents a pronounced leap in the de novo design and in cellulo engineering of artificial metalloenzymes, paving the way for abiological catalysis in living systems.
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Nov 2025
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I04-Macromolecular Crystallography
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Pyung-Gang
Lee
,
Linxiang
Yin
,
Xin
Wei
,
Jingyuan
Shi
,
Geoffrey
Masuyer
,
Travis G.
Wentz
,
Pengsheng
Chen
,
Ying
Xu
,
Junjie
Liang
,
Haonan
Zhang
,
Sara
Persson Kosenina
,
Briallen
Lobb
,
Michael
Mansfield
,
Sarjeet S.
Gill
,
Sabine
Pellett
,
Pal
Stenmark
,
Andrew C.
Doxey
,
Min
Dong
Open Access
Abstract: Insecticidal bacterial proteins play key roles in insect-bacteria interactions and have been used as biopesticides. Here, we identify two insecticidal proteins in Paeniclostridium ghonii, designated PG-toxin 1 (PG1) and PG-toxin 2 (PG2), which are homologs of botulinum neurotoxins (BoNTs). Unlike BoNTs, PG1 and PG2 contain two separate proteins: One is the protease light chain (LC), and the other is the heavy chain containing the translocation domain and the receptor binding domain. Crystal and cryo–electron microscopy structures show a conserved BoNT-like architecture but without an interchain disulfide bond. Functional characterizations establish that the LCs of PG1 and PG2 cleave insect synaptosomal–associated protein 25 (SNAP25), but not human or rat SNAP25, and microinjection of PG1 and PG2 caused paralysis and death in Drosophila and Aedes mosquitoes. These findings identified unique two-component BoNT-like insecticidal proteins, revealing insights into the evolution of the BoNT family of toxins, and broadening our understanding of bacteria that can be used for biopest controls.
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Nov 2025
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