Toshiya Senda

Understanding the structural properties of pathogenic viruses in their native, infectious state is critical for rational vaccine and drug development. However, conducting cryo‐electron microscopy (cryo‐EM) under biosafety level 3 (BSL‐3) containment presents significant challenges. Here, we established and validated a high‐end cryo‐EM workflow within a BSL‐3 facility, comprehensively integrating facility design, containment measures, sample handling, cryo‐transfer systems, and decontamination protocols. To demonstrate the utility and reproducibility of this workflow, we applied cryo‐electron tomography to investigate severe acute respiratory syndrome coronavirus 2 in its native, active state. Our observations confirm that unfixed viral particles display pleomorphic features comparable to those previously reported in fixed or inactivated preparations, validating that these structural characteristics represent native features rather than artifacts of chemical fixation. Furthermore, spatial redistribution of surface spikes was observed. The establishment of this robust BSL‐3 cryo‐EM workflow provides a practical blueprint for the structural characterization of emerging high‐consequence pathogens, supporting pandemic preparedness, vaccine development, and antiviral discovery.

In cancer cells, Ras protein mutations activate a signaling cascade that phosphorylates kinases, transcription, and translation factors, driving cancer cell proliferation. One such factor, FOXO3a, promotes apoptosis-related gene transcription. However, in many cancer cells, FOXO3a is phosphorylated and is bound to 14-3-3ζ at phosphorylation sites. The 14-3-3ζ binding displaces phosphorylated FOXO3a from DNA, suppressing apoptosis. Since the phosphorylation sites are far from the DNA-binding domain (DBD) of FOXO3a, the mechanism of displacement remains unclear. Using isothermal titration calorimetry and fluorescence-detection size-exclusion chromatography, we find that 14-3-3ζ strongly displaces DNA from di-phosphorylated FOXO3a (dpFOXO3a), despite similar dissociation constants for dpFOXO3a-14-3-3ζ and dpFOXO3a-DNA. Nuclear magnetic resonance data identify weak, but direct binding of 14-3-3ζ to the DBD, suggesting direct competition. These findings suggest that 14-3-3ζ enhances its competitive ability by dual tethering to the DBD of FOXO3a via phosphorylation sites, effectively displacing DNA.

X-ray crystallography remains a powerful technique for determining high-resolution protein structures; however, obtaining high-quality crystals is a significant bottleneck. This study presents a detailed experimental workflow that employs differential scanning fluorimetry (DSF) to optimize protein crystallization. DSF, which measures protein thermal stability, was used to refine both protein buffer composition and crystallization conditions. The method was applied to two distinct proteins: CreD, a nitrosuccinate lyase, and HIRA, a histone chaperone. For CreD, DSF-based optimization of the protein buffer enhanced the crystal quality, increasing the resolution from 3.32 to 2.18 Å. For HIRA(644–1017), DSF-guided optimization of the protein buffer significantly improved the protein solubility from 0.1 to 19.1 mg ml ⁻¹ , facilitating the growth of initial crystals. Further optimization of the crystallization conditions using DSF, combined with microseeding, improved the crystal quality, leading to structure determination at 2.45 Å resolution. This study demonstrates that DSF is a valuable tool for efficiently optimizing protein crystallization. The workflow presented here, involving initial DSF-based optimization of protein buffers followed by DSF-guided optimization of crystallization conditions, offers a rational approach to enhancing protein crystal quality, thereby facilitating structure determination by X-ray crystallography.

Rickets, a disorder of bone formation, was originally known as nutritional rickets due to vitamin D deficiency. Advances in science have since identified various genetic forms, typically involving loss‐of‐function mutations in vitamin D activation or other mineral metabolism pathways. Recently, type 3 rickets was identified as a previously undescribed gain‐of‐function mutation in CYP3A4 (Ile301Thr). This mutant enzyme leverages the unique features of cytochrome P450 to produce an inactive vitamin D metabolite, 11α,25(OH) ₂ D ₃ , resulting in insufficient active vitamin D. The discovery of this unique gain‐of‐function aetiology and its associated metabolite opens a significant new direction in rickets research.

A densely pyrene-modified protein nanocage enables aqueous dispersion of the solvatochromic dye Nile Red. On paper, the solution exhibits a reversible blue–purple color shift upon heating and cooling, reflecting changes in local water content.

Guanosine triphosphate (GTP) is increasingly recognized as a critical actor in cancer cell proliferation, yet its regulatory mechanism remains incompletely defined. A key contributor to elevated GTP levels in tumors is inosine monophosphate dehydrogenase 2 (IMPDH2), a rate‐limiting enzyme in the de novo guanine nucleotide biosynthetic pathway. Although IMPDH inhibitors, mycophenolic acid (MPA) and mycophenolate mofetil (MMF), have shown potential in cancer therapies, their success has been limited due to their immunosuppressive side effects and several unresolved regulatory mechanisms, including paradoxical control of IMPDH activity by GTP. This review provides a systematic summary of the current understanding of IMPDH biology, emphasizing its complex regulation and therapeutic relevance in cancer. We will outline key unresolved questions, including isozyme‐specific roles and mechanisms for escaping regulation, and propose mechanistic and translational strategies to design IMPDH‐targeted cancer therapies.

Modular polyketide synthases (PKSs) are large multi-domain enzymes critical for the biosynthesis of polyketide antibiotics. However, challenges with structural analysis limits our mechanistic understanding of modular PKSs. In this report, we explore the potential of ancestral sequence reconstruction (ASR) for structure analysis of target proteins. As a model, we focus on the FD-891 PKS loading module composed of ketosynthase-like decarboxylase (KS_Q), acyltransferase (AT) and acyl carrier protein (ACP) domains. We construct a KS_QAncAT chimeric didomain by replacing the native AT with an ancestral AT (AncAT) using ASR. After confirming that KS_QAncAT chimeric didomain retains similar enzymatic function to the native KS_QAT didomain, we successfully determine a high-resolution crystal structure of the KS_QAncAT chimeric didomain and cryo-EM structures of the KS_Q-ACP complex. These cryo-EM structures, which could not be determined for the native protein, exemplify the utility of ASR to enable cryo-EM single-particle analysis. Our findings demonstrate that integrating ASR with structural analysis provides deeper mechanistic insight into modular PKSs. Furthermore, applying ASR to a partial region of the targeted multi-domain proteins could expand the potential of ASR and may serve as a valuable framework for investigating the structure and function of various multi-domain proteins.