Structure

In this issue of Structure, Hylton et al.1 present MOSAIC, an accessible, acquisition-software-agnostic framework for montage cryo-electron tomography (cryo-ET). By streamlining acquisition and stitching workflows, MOSAIC lowers technical barriers to wide-field imaging, maximizing data recovery from cryolamellae and making meso-scale cellular architecture broadly accessible across diverse cryo-ET platforms.

Candida auris is an emerging multidrug-resistant fungal pathogen responsible for severe healthcare-associated infections worldwide; yet, the molecular architecture of its translational apparatus remains poorly understood. Here, we present a structural and functional analysis of the C. auris ribosome. Using in-house rRNA sequencing, we found that C. auris rRNA displays substantial divergence, including the lowest 18S rRNA identity among the pathogenic Candida species examined and an unusual substitution at a functionally important nucleotide in helix h31 of the small subunit P-site region. To define the structural basis of these differences, we determined single-particle cryo-electron microscopy structures of the vacant C. auris 80S ribosome and its complexes with cycloheximide (CHX), blasticidin S (BLS), and geneticin (G418). Together with cell-free translation inhibition assays, these results establish a structural framework for understanding the C. auris ribosome and reveal how local ribosomal variation within a conserved fungal scaffold can be associated with species-specific inhibitor responses.

In a recent issue of Nature Chemical Biology, Nguy et al.1 report the discovery of an array of cytochrome P450 enzymes that challenge the long-standing view that P450 hemes must be coordinated by a cysteine thiolate. Many of these "noncanonical" P450s instead use serine or selenocysteine ligands. The characterization of two selected variants revealed distinct structural and spectroscopic features.

Class I membrane fusion proteins from many important viruses have been structurally characterized at multiple stages during the fusion process. Their fusion pathways have largely been inferred from the trimeric architectures captured throughout this dynamic transition. Here, a class I baculoviral F protein, that forms patches of hexagonal lattices on the viral membrane was structurally characterized to analyze its fusion dynamics. The pre-fusion and post-fusion ectodomains of this F protein adopt trimeric conformations, similar to those of pneumovirus F proteins. A metastable ectodomain dimer was captured during the pre-fusion to post-fusion transition, coinciding with a relocation of the fusion peptide from its sequestered position within the central chamber of the pre-fusion trimer. This process of trimer dissociation and reassembly may explain how fully buried fusion peptides become exposed to enable subsequent membrane fusion.

MRP2 (ABCC2) contributes to multidrug resistance in cancer treatment and plays a key role in liver detoxification. Recent studies on mammalian MRP2 have elucidated its basic structure and regulation via R-domain phosphorylation and displacement, but have not yet explained its substrate preferences or substrate/inhibitor binding modes. We conducted structural and functional studies on human MRP2 with its substrate SN-38G or inhibitor MK-571. Our research provides structural insight into substrate recognition by MRP2 and the positive cooperativity observed in ATP hydrolysis upon SN-38G stimulation, and reveals structural features that contribute to the recognition of glucuronidated substrates. We further identify an MK-571-associated density within the substrate-binding region rather than the nucleotide-binding sites, although its precise binding mode remains unresolved.

In this meet-the-author Q&A, we speak to Stefan Raunser from the Max Planck Institute of Molecular Physiology in Dortmund about his research group’s recent Structure paper entitled “Streamlined montage cryo-electron tomography for exploring the ultrastructure of cells and tissues,” his work, and his career.

The maturation of key coagulation factors requires γ-carboxylation catalyzed by γ-glutamyl carboxylase (GGCX), in which vitamin K hydroquinone (VKH2) is oxidized to vitamin K epoxide (VKO) and recycled by vitamin K epoxide reductase (VKORC1). Clinically, vitamin K antagonists (VKAs) inhibit VKORC1 but are thought not to target GGCX. Here, we demonstrate that a member of VKA, anisindione and its analogs can dock within the VK-binding pocket of GGCX. Importantly, both in vitro and cell-based γ-carboxylation assays showed that anisindione inhibits GGCX activity. Furthermore, our cryo-electron microscopy structure of the GGCX-BGP (bone Gla protein or osteocalcin)-anisindione complex reveals that anisindione directly occupies the VK-binding pocket of GGCX, consistent with competitive inhibition with VK. These results establish anisindione as a structural prototype for direct GGCX inhibition and provide a structural framework for developing anticoagulants beyond VKORC1.

The 3D structure and mechanism of action are unknown for the integral plasma membrane transport protein solute carrier 4A10, which has been characterized functionally as an electroneutral Na+:HCO3- cotransporter. We used structure prediction and molecular dynamics simulations to study the binding of the transported ions to the solute carrier 4A10 protein and suggest a model of sequential binding of Na+ followed by HCO3- to the ion binding domain. The binding of HCO3- to the protein appears to depend absolutely on Na+ binding. Conversely, the binding of HCO3- stabilizes the interaction between Na+ and its binding site. Measurements of intracellular pH and Na+ concentration revealed the dependence of Na+ on HCO3- transport. The study lays the necessary foundation for targeted experimental analysis of ion translocation and for the development of selective transport inhibitors of solute carrier 4A10 and probably other proteins of the protein family of HCO3- transporters.

Gcn5-related N-acetyltransferases (GNATs) are considered a "megafamily" that ranks among the most structurally and sequentially diverse superfamilies in the CATH (Class, Architecture, Topology, Homology) database. In this vast superfamily, several types of protein functions have been explored throughout evolution, yet the evolutionary pathways that led to such diversity remain poorly understood. To investigate these concepts further, we selected a functionally distinct GNAT subgroup called polyamine N-acetyltransferases (PAATs). These enzymes acetylate polyamines that are crucial for cellular homeostasis. While PAATs from different domains of life catalyze the same reaction, their residue conservation patterns, oligomeric states, and presence of allosteric sites vary. Despite their biological importance, many putative PAATs remain uncharacterized, limiting our ability to infer evolutionary relationships, understand how functional properties emerged, and appreciate the extent of their structural diversity and substrate specificity. Here, we present a characterization of a large subset of PAAT enzymes, including their likely oligomeric states, functional site properties, and experimental functions.

Retrograde trafficking from endosomes to trans-Golgi network is essential for cellular homeostasis. While the WDR11-FAM91A1-C17orf75 (WFC) complex facilitates this process, its structural organization and the role of C17orf75 remain unclear. Here, we present cryo-EM structures of human WFC complex in monomeric and dimeric forms at 3.34 Å and 3.07 Å resolution, respectively. The WFC complex forms a dimer through the α-solenoid domains of WDR11. FAM91A1 serves as a central scaffold that interacts directly with both WDR11 and C17orf75, whereas WDR11 and C17orf75 have no direct contact. C17orf75 features an N-terminal longin-like domain and a C-terminal DENN-like domain and contains a positively charged groove that may cooperate with WDR11 in recognizing acidic-cluster-containing cargo proteins. Functional analyses demonstrate that C17orf75 is indispensable for the proper intracellular localization of the acidic-cluster-containing cargo proteins CI-MPR, KIAA0319L, and VAMP4. These findings establish C17orf75 as an integral component of the WFC complex in endosome-to-TGN trafficking.

In this meet-the-author Q&A, we speak to Blake Wiedenheft from Montana State University about his research group's recent Structure paper entitled "Identification and structure determination of a type III-Bv CRISPR complex that post-translationally modifies an associated toxin" and his work and career.

In this Voices article, participants from the recent wwPDB Workshop on Fragment Screening at the EMBL-EBI and their colleagues discuss recent advances, current challenges, and future opportunities in crystallographic and NMR fragment screening and describe how the PDB is adapting to support this rapidly evolving field.

In this issue of Structure, Puri et al.1 show that N-glycosylation stabilizes antibody light chains that are associated with systemic amyloidosis. The association between a stabilizing post-translational modification and a disease driven by protein misfolding and aggregation is counter-intuitive, highlighting the complexity of this rare but often fatal disorder.

In this issue of Structure, Zakrzewska et al.1 reveal the molecular principles underlying how a wide variety of natural and synthetic paralytic shellfish toxins (PSTs) bind to saxiphilins, naturally occurring proteins found in certain frog species, providing crucial insights into PST interactions with biological targets more broadly.

Acetyl-coenzyme A synthetases convert ATP, acetate, and coenzyme A (CoA) into acetyl-CoA, a central metabolite that fuels lipid biosynthesis and regulates protein and RNA acetylation. ACS enzymes contain N- and C-terminal domains that coordinate a two-step ping-pong mechanism involving sequential adenylation and thioester formation at the interdomain interface. How domain motions coordinate these chemical steps remains unclear. Here, we report single-particle cryo-electron microscopy structures of Schizosaccharomyces pombe ACSA captured in apo, pre-adenylation, intermediate, and product states. These structures reveal ligand-dependent reorganization of the C-terminal domain: apo and pre-adenylation forms display increased conformational heterogeneity, whereas intermediate- and product-bound states adopt ordered conformations compatible with catalysis. Structure-guided mutagenesis and in vitro activity assays, together with sequence conservation, support the functional importance and evolutionary conservation of the observed conformational transitions across ACS homologs. These findings establish a ligand-coupled interdomain rearrangement mechanism underlying catalysis by ACS enzymes and a structural framework for inhibitor development.

Septal peptidoglycan (sPG) biosynthesis during bacterial cell division is driven by the dynamic divisome complex. Its core components, glycosyltransferase FtsW and transpeptidase FtsI are responsible for glycan chain polymerization and crosslinking, respectively. FtsI is also the target of β-lactams. The essential membrane complex FtsQ-FtsB-FtsL regulates FtsWI enzymatic activity. However, the mechanism of FtsQBLWI-mediated sPG synthesis and β-lactam-induced conformational changes have remained elusive. Here, we present cryo-electron microscopy (cryo-EM) structures of the Pseudomonas aeruginosa FtsQBLWI complex in the apo state and bound to aztreonam or imipenem. Our work reveals intricate structural details, including the putative substrate-binding cavities of FtsW, FtsI-mediated allosteric activation of FtsW, and β-lactam-triggered conformational rearrangements. Collectively, these structural, genetic and biochemical analyses reveal the mechanism of FtsQBLWI-controlled sPG synthesis and β-lactam action on this complex, providing a molecular basis for optimizing existing β-lactams and developing novel antibiotics.

Polyamines, well-known regulators of the mitochondrial calcium (Ca2+) uniporter channel, show unexpected effects when binding the channel from within the matrix. Using cryo-EM, molecular dynamics simulations, and mutagenesis experiments, we determine that polyamines achieve such regulation by binding within the pore to a ring of negative residues forming a matrix gate, inhibiting Ca2+ conduction. In whole-mitoplast electrophysiology assays, matrix polyamines cause a gradual increase in Ca2+ currents during prolonged conduction, due to relief of this inhibition. Notably, this electrostatic binding increases 3-fold as the inner membrane depolarizes, preventing Ca2+ efflux. Additionally, we also identify that phospholipids form part of the Ca2+ conduction pathway through MCU. Because we find significant variability in matrix polyamine content across mouse organs, this unexpected mechanism for sculpting the mitochondrial Ca2+ waveform suggests a tissue-specific regulation of metabolism.

Mitochondrial architecture plays a critical role in cellular function, yet how organelle structure, metabolic density, and subcellular position are jointly remodeled across whole cells remains poorly understood. We applied quantitative 3D soft X-ray tomography to analyze intact INS-1E cells in a native, cryo-hydrated state. By integrating morphometric profiling with voxel-level linear absorption coefficients (LACs) and contour-based radial mapping, we tracked the structural, biochemical, and spatial remodeling of fragmented, intermediate, and interconnected mitochondrial morphotypes under high glucose and Exendin-4 stimulation. High glucose induces morphotype-specific hypertrophy, fission, and perinuclear redistribution of low-density fragments. Co-stimulation with Exendin-4 stabilizes interconnected networks and increases metabolic density at the cell periphery. Morphotype-resolved analysis uncovers a structure-density-location coupling in which mitochondrial shape, macromolecular packing, and radial position shift in concert. These results offer a quantitative framework and high-resolution spatial constraints for whole-cell modeling of organelle dynamics.

T cell receptors (TCRs) specific for cancer neoantigens are important for anti-tumor immunity and immunotherapy. To understand the structural basis for T cell recognition of cancer neoantigens, we studied oligoclonal TCRs from patients with melanoma that recognize a neoepitope arising from a driver mutation in NRAS (NRASQ61K) presented by HLA-A1. Structures of these TCRs in unbound form and bound to NRASQ61K-HLA-A1 revealed that they employ chemically distinct strategies and engagement modes to distinguish between mutant and wild-type NRAS. The structures explain how the NRASQ61K mutation rendered a self-antigen visible to T cells. We additionally benchmarked AlphaFold-based modeling of these complexes, showing that predictive accuracy varies markedly across TCR-peptide-MHC targets. We found that conformational plasticity can dramatically impact complex assembly accuracy. These findings define the basis for TCR recognition of a cancer neoantigen and provide stringent tests for computational modeling of TCR-peptide-MHC interactions relevant to cancer immunotherapy.

Caspase-4 drives non-canonical inflammasome signaling by cleaving gasdermin D (GSDMD) to trigger pyroptosis. Cleavage of the interdomain linker (IDL) in caspase-4 yields distinct autoprocessed forms—p20/p12, p22/p10, and p20/p10. While both p22/p10 and p20/p10 forms of caspase-4 are capable of processing GSDMD, how these GSDMD-cleaving states are structurally organized in complex with full-length human GSDMD remains unclear. Here, we present cryo-EM structures of full-length human GSDMD bound to two human caspase-4 autoprocessed forms, p22/p10 and p20/p10. Both complexes preserve exosite-mediated recognition of the GSDMD C-terminal domain, but they display distinct catalytic-groove occupancy. In the p22/p10 complex, a residual LEED-containing IDL segment folds back into the catalytic pocket, whereas in the p20/p10 complex, the GSDMD FLTD cleavage-site linker occupies the same groove. These structures reveal how distinct IDL-processing states are associated with different modes of catalytic-groove occupancy and provide a structural framework for understanding full-length GSDMD recognition by human caspase-4.