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Abstract: Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) target proteins for degradation by co-opting an E3 ligase. While heterotrivalent PROTACs that can recruit multiple E3 ligases have been described, all MGDs reported to date depend on a single E3. Using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can recruit CUL4DCAF16 and CRL1FBXO22 in parallel to degrade SMARCA2/4. Deep mutational scanning identifies C173 in DCAF16 as essential for degrader activity and intact protein mass spectrometry confirms covalent modification at this site. Elucidating the ternary complex structure reveals a unique binding mode and a distinct interface of neointeractions that underlie degrader specificity. We demonstrate that ligase dependency is chemically and genetically tunable. Minimal compound modifications shift preference from DCAF16 to FBXO22, while a single substitution boosts degrader dependency on DCAF16. These results establish a framework for designing tunable dual E3 ligase degraders to mitigate potential resistance mechanisms.

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Abstract: Type IV pili are long, filamentous structures that extend from bacterial cell surfaces, enabling cells to respond to changing environments and facilitating genome plasticity. Thermus thermophilus HB27 produces two different type IV pili, each exhibiting distinct structural and functional properties. Here, we combine cryo-electron tomography, mutagenesis, and AlphaFold predictions to generate hypothetical in situ models of the T. thermophilus type IV pilus assembly machinery. Using single-particle cryo-electron microscopy, we determine structures of both filament types, enabling modelling of their surface glycans. Molecular dynamics simulations further reveal the flexibility of these glycans on extrusion. Integration of the filament structures with our hypothetical model of the assembly machinery offers a framework for further dissecting T4P architecture and biogenesis.

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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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Abstract: Chaperonins are essential protein-folding machines, classified into three structural and phylogenetic groups: Group I (bacterial GroEL), Group II (archaeal thermosome and eukaryotic CCT), and Group III (bacterial thermosome-like). Using ancestral sequence reconstruction (ASR) and protein resurrection, we inferred and experimentally characterized the last common ancestors of these groups (Ancestral Chaperonins ACI, ACII, and ACIII). The resurrected proteins exhibited ATPase activity (except ACII) and protected client proteins from heat-induced inactivation. Structural analyses by electron microscopy and Cryo-EM revealed that ACI forms single 7-mer rings, whereas ACII adopts a mixed population of single/double 8-mer rings, representing the first experimental observation of intermediate oligomeric states. ACII also features a unique cochaperonin-independent closure mechanism, distinct from modern Group I and II chaperonins. Together, these findings provide the experimental structural reconstruction of the most ancient and complex multimeric proteins so far, uncover novel intermediate states in chaperonin evolution, and offer a direct empirical framework for studying the emergence of multimeric complexity in early cellular life.

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Abstract: High-resolution structural studies have mainly focused on two out of the six adenovirus genera: mastadenoviruses and atadenoviruses. Here we report the high-resolution structure of an aviadenovirus, the poultry pathogen fowl adenovirus serotype 4 (FAdV-C4). FAdV-C4 virions are highly thermostable, despite lacking minor coat and core proteins shown to stabilize the mast- and atadenovirus particles, having no genus-specific cementing proteins, and packaging a 25% longer genome. Unique structural features of the FAdV-C4 hexon include a large insertion at the trimer equatorial region, and a long N-terminal tail. Protein IIIa conformation is closer to atadenoviruses than to mastadenoviruses, while protein VIII diverges from all previously reported structures. We interpret these differences in light of adenovirus evolution. Finally, we discuss the possible role of core composition in determining capsid stability properties. These results enlarge our view on the structural diversity of adenoviruses, and provide useful information to counteract fowl pathogens or use non-human adenoviruses as vectors.