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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: Cyclin-dependent kinases (CDKs) are prototypical regulators of the cell cycle. The CDK-activating kinase (CAK) acts as a master regulator of CDK activity by catalyzing the activating phosphorylation of CDKs on a conserved threonine residue within the regulatory T-loop. However, structural data illuminating the mechanism by which the CAK recognizes and activates CDKs have remained elusive. Here, we determine high-resolution structures of the CAK in complex with CDK2 and CDK2-cyclin A2 by cryogenic electron microscopy. Our structures reveal a T-loop–independent kinase-kinase interface with contributions from both kinase lobes. Computational analysis and structures of CAK in complex with CDK1-cyclin B1 and CDK11 indicate that these structures represent the general architecture of CAK-CDK complexes. These results advance our mechanistic understanding of cell cycle regulation and kinase signaling cascades.

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Abstract: CDK7 has emerged as a cancer target because of its pivotal roles in cell cycle progression and transcription. Several CDK7 inhibitors (CDK7i) are now in clinical evaluation. Identifying patients most likely to respond to treatment and early detection of tumour evolution towards resistance are necessary for optimal implementation of cancer therapies. Continuous culturing of prostate cancer cells with Samuraciclib, a non-covalent ATP-competitive CDK7i, led to outgrowth of resistant cells. These were characterised by the acquisition of a single base change in the CDK7 gene, Asp97 to Asn (D97N). Mutant cells were resistant to other non-covalent CDK7i but remained sensitive to covalent CDK7i. Cryo-EM structure and kinase ligand affinity determinations revealed reduced affinity of the CDK7-D97N mutant for non-covalent CDK7i. Remarkably, Asp97 is absolutely conserved in human CDKs, inferring its importance for the activities of all CDKs. Consistent with this, mutation of the homologous residue in CDK12 (D819N) or CDK4 (D99N) promoted resistance to drugs that inhibit these CDKs. Our findings reveal a general mechanism for acquired resistance with obvious implications for patients treated with CDK inhibitors.

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Abstract: SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2’ cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2’ cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike’s stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.