As 2025 comes to an end, we want to highlight some of the rising young faculty members who have published their exciting work from different areas of structural biology in Structure this year. We have asked them to tell us more about their interests, careers, and research programs.

Deep-learning models have transformed structural biology by enabling reliable prediction of protein 3D structure models and providing confidence metrics such as predicted local distance difference test (pLDDT) to estimate local uncertainties. However, whether pLDDT reflects intrinsic protein flexibility remains unclear. Defining and quantifying flexibility and protein dynamics through experiments and computation is essential for advancing our ability to model and interpret conformational changes across different timescales.

In this issue of Structure, Henriques et al. present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.

The microtubule-associated protein tau is implicated in neurodegenerative diseases characterized by amyloid formation. Mutations associated with frontotemporal dementia increase tau aggregation propensity and disrupt its endogenous microtubule-binding activity. However, the structural relationship between aggregation propensity and biological activity remains unclear. We employed a multi-disciplinary approach, including computational modeling, NMR, cross-linking mass spectrometry, and cell models to engineer tau sequences that modulate its structural ensemble. Our findings show that substitutions near the conserved "PGGG" β-turn motif informed by tau isoform context reduce tau aggregation in vitro and can counteract aggregation from disease-associated proline-to-serine mutations. Engineered tau sequences maintain microtubule binding and explain why 3R isoforms exhibit reduced pathogenesis compared to 4R. We propose a simple mechanism to reduce the formation of pathogenic tau species while preserving biological function, thus offering insights for therapeutic strategies aimed at reducing tau protein misfolding in neurodegenerative diseases.

Vibrio cholerae cytolysin (VCC) is a β-barrel pore-forming toxin (β-PFT). The membrane insertion of its pore-forming "pre-stem" motif is the most crucial step in the pore-formation mechanism. In the soluble monomeric form, pre-stem remains clamped against the central cytolysin domain by the so-called cradle loop. In the course of oligomeric pore-formation in the target membranes, the cradle loop gets detached from the pre-stem and reorients, thus allowing the pre-stem to extend and insert into the membrane. Here, we show that the specific cradle loop residue(s) play crucial roles in governing the pore-formation mechanism of VCC by establishing decisive interactions with the neighboring structural domains/modules. The alteration of the cradle loop residue, Y194 in particular, compromises the membrane-insertion of the pre-stem, and tends to arrest the membrane-bound toxin in the pre-pore-like oligomeric states. Our study suggests that the native cradle loop architecture, with its intact contacts with the surrounding interaction partners, is essential for VCC pore-formation.

Memory is incorporated into the brain as physicochemical changes to engram cells. These neuronal populations form complex neuroanatomical circuits, are modified by experiences to store information, and allow memory recall. At the molecular level, learning modifies synaptic communication to rewire engram circuits. How macromolecules are organized within engram synapses is unknown. Here, we establish engram labeling technology combined with cryogenic correlated light and electron microscopy (cryoCLEM)-guided cryogenic electron tomography (cryoET) to visualize the in-tissue 3D macromolecular architecture of engram synapses of a contextual fear memory within the mouse hippocampus. Engram synapses exhibited structural diversity of macromolecular constituents and organelles in both pre- and postsynaptic compartments and within the synaptic cleft, including in membrane proteins, synaptic vesicle occupancy, and F-actin copy number. This "engram to tomogram" approach, harnessing in vivo functional neuroscience and structural biology, provides a methodological framework for testing fundamental molecular plasticity mechanisms within engram circuits.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is central to energy metabolism by coupling NADH oxidation and quinone reduction with proton translocation across the membrane. Electrons are transferred from the primary acceptor flavin mononucleotide via a chain of iron-sulfur clusters to quinone. The enigmatic cluster N1a is conserved, but not part of this electron transfer chain. We reported on variants of the complex in which N1a is not detectable by EPR spectroscopy. This was tentatively attributed to the lower redox potential of the variant N1a. However, it remained an open question, whether the variants contain this cluster at all. Here, we determined the structures of these variants by X-ray crystallography and cryogenic-electron microscopy. Cluster N1a is present in all variants and the shift of its redox potential is explained by nearby structural changes. A role of the cluster for the mechanism of the complex is discussed.

Aerobic anoxygenic phototrophic bacteria (AAPB) are widely distributed in nature and they are important members of the marine phototrophic community. However, a structural and functional understanding of the AAPB photosynthetic apparatus is still lacking. Here, we present cryo-EM structures of the LH1-RC (core) and LH2 (peripheral) photocomplexes from the model aerobic phototroph Erythrobacter (Ery.) sanguineus. The LH1 αβ-heterodimers bind the carotenoids bacteriorubixanthinal and caloxanthin-pigments that are absent from anaerobic anoxygenic phototrophs-to form a closed ring structure. Ery. sanguineus LH1-RC contains a lipid-anchored polypeptide unrelated to any of the auxiliary proteins identified in the core complexes of purple bacteria so far. The Ery. sanguineus LH2 complex shows unique absorption characteristics, with its Q transition being blue-shifted to 814 nm. This work provides structural insights into the unusual photosynthetic properties of AAPB and points to new avenues to further explore their biology.

Sulfate-reducing bacteria import organosulfur compounds from the environment for anaerobic respiration. They contribute to human disease and are problematic in industrial settings because they produce hydrogen sulfide. Here, we demonstrate how the sulfate-reducing bacterium Oleidesulfovibrio alaskensis imports isethionate, a common organosulfonate, using a tripartite ATP-independent periplasmic (TRAP) transporter (OaIsePQM). The cryo-EM structure of isethionate-bound OaIseQM to 2.98 Å resolution defines the substrate-binding site, two Na-binding sites, and a distinct fusion helix. Key residues within the OaIseQM substrate-binding site are identified using substitution and proteoliposome assays. Functional studies demonstrate that OaIseQM requires the substrate-binding protein (OaIseP) and a Na gradient to drive transport. Modeling of the OaIsePQM complex supports that elevator-type conformational changes are involved in this unique coupled transport process. This work expands our knowledge of the transport of organosulfur compounds in bacteria and establishes OaIsePQM as a new model system for exploring the mechanism of TRAP transporters.

In this issue of Structure, Deshmukh et al. reveal that β cells actively remodel insulin secretory granules in response to specific physiological cues, altering granule density, proinsulin processing, and spatial distribution. This stimulus-specific structural maturation highlights how β cells sculpt their secretory machinery, offering new insights into insulin release regulation.

Inherited mutations in VPS35 and LRRK2 kinase lead to hyperphosphorylation of Rab GTPases. RH2 domain-containing proteins from the RILP homology family, such as RILPL1, are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8A and phospho-Rab10. Phospho-Rabs are also seen on lysosomal membranes in complex with RILPL1 and TMEM55B, a 284-residue lysosomal membrane protein lacking homology to known proteins. Here, we report crystal structures of the cytosolic region 80-166 of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, which we define as the TMEM55B-binding motif (TBM). The RILPL1 TBM sits in a shallow groove across two tandem RING-like domains of TMEM55B, each forming a Zn-stabilized 40-residue β-sandwich. Co-immunoprecipitation and mass spectrometry studies indicate that TMEM55B forms complexes independently of phospho-Rabs with conserved TBMs found in JIP3, JIP4, OCRL, WDR81, and TBC1D9B. These studies suggest that TMEM55B acts as a central hub for adaptor recruitment on lysosomes.

Accurate model building in intermediate-resolution cryo-EM maps normally requires flexible fitting of reliable initial structures. However, while deep learning-based methods such as AlphaFold2 can predict highly accurate structures, the predicted structures often differ from experimental EM maps on both global and local scales, which poses a great challenge to accurate model building in intermediate-resolution EM maps with such initial structures. Addressing the challenge, we propose CryoEvoBuild, an automated method for improved protein model building from intermediate-resolution EM maps through the effective integration of evolutionary and experimental information. CryoEvoBuild implements a novel domain-wise fitting, refinement, assembly, and rebuilding pipeline with a recycling framework guided by AlphaFold2. Extensive benchmarking on a diverse test set of 117 maps at 4.0-10.0 Å resolutions demonstrates that CryoEvoBuild significantly improves the accuracy of AF2-predicted structures and outperforms state-of-the-art approaches, including EMBuild and phenix.dock_and_rebuild.

Sacituzumab govitecan (SG) is a therapeutic antibody-drug conjugate globally approved for the treatment of breast cancer. SG targets the trophoblast cell-surface antigen-2 (Trop2) at the surface of cancer cells to deliver the cytotoxic topoisomerase I inhibitor SN-38 to the tumor microenvironment. SN-38 is covalently linked to the humanized monoclonal antibody (mAb) sacituzumab via a hydrolyzable linker. Here, we describe the 1.56-Å X-ray crystal structure and stoichiometry of the human Trop2 ectodomain in complex with a sacituzumab (hRS7) antigen-binding Fab fragment. The complex reveals a 2:2 stoichiometry where two sacituzumab Fabs bind across the two Trop2 dimer subunits, inducing a conformational change compared to the apo-structure. Cryo-electron microscopy (cryoEM) and size-exclusion chromatography in combination with multi-angle light scattering (SEC-MALS) analysis of the intact sacituzumab mAb bound to the Trop2 ECD reveals a complex whereby sacituzumab engages two Trop2 dimers in a 2:4 stoichiometry.

Podophage tails are too short to traverse the cell envelope and require internal core proteins to assemble into a transmembrane channel for genome delivery during infection. However, high-resolution structures of near-complete cores remain scarce. Here, we present the near-atomic-resolution cryo-electron microscopy (cryo-EM) structure of the drug-resistant E. coli phage E1004, which features a T7-like core-portal-tail structure with six P22-like tailspikes. We found that the cylindrical core comprises four proteins: gp17, gp27, gp28, and gp29. Gp29 forms a tetramer, while gp28 and gp27 assemble into octamers. Notably, there are sixteen copies of gp17 in two conformations, distinct from the small core protein gp6.7 in T7. The gp17-gp27 complex reveals the mechanism for mediating the symmetry adjustment at the core-portal interface. Moreover, comparative analysis with other podophage cores highlights diversity in core protein composition and organization, particularly among the small core proteins. We propose that these variations represent evolutionary adaptations to diverse host envelopes.

In a recent issue of Nature Structural & Molecular Biology, Anton et al. produce the first in situ visualization of Plasmodium falciparum ribosomes within infected erythrocytes. Using cryoelectron tomography and cryoelectron microscopy, ten ribosomal states are resolved, five previously unseen in eukaryotes, providing a more comprehensive parasite translation elongation cycle. The work describes parasite-specific translation dynamics, showing how the antimalarial cabamiquine disrupts elongation.

Why do certain drugs bind human OAT1 with much higher affinity than the rat ortholog? In this issue of Structure, Jeon et al. reveal that serine 203, which is present only in human OAT1, coordinates with a chloride ion and this S203-chloride interaction is crucial for the high-affinity binding of olmesartan and other drugs.

HBV causes chronic infections that can lead to severe liver disease, yet current treatments rarely achieve a cure. The HBV capsid is a critical therapeutic target, but structural insights have largely relied on E. coli-derived particles lacking native modifications. Here, we present near-atomic resolution cryo-electron microscopy (EM) structures of HBV capsids purified from human embryonic kidney (HEK-293T) cells, capturing authentic architecture and post-translational modifications. A hydrophobic pocket at the intradimer interface harbors lipid-like densities corresponding to stearic and palmitic acids, confirmed by gas chromatography-mass spectrometry. Molecular dynamics simulations revealed that pocket accessibility is regulated by rotamer states of Lys96, Phe97, and Gln99, supporting an induced fit model of fatty acid binding. Reduced phosphorylation and increased RNA content further modulate capsid conformation and pocket openness. These findings highlight the dynamic regulation of HBV capsid structure and provide a framework for understanding how capsid conformational dynamics contribute to viral assembly and envelopment.

Insights into bacterial metabolic adaptation during stress is crucial for understanding early mechanisms of antibiotic resistance. In the Gram-negative bacterium Escherichia coli, the universal stringent response produces the alarmones (p)ppGpp that target many cellular proteins. The cellular nucleosidase PpnN is regulated by (p)ppGpp and was shown to balance bacterial fitness and persistence during fluoroquinolone exposure. pppGpp and ppGpp both activate PpnN, but differentially regulate its cooperativity via an unknown mechanism; furthermore, the catalytic mechanism of PpnN has remained unclear. Here, we provide mechanistic insights into the interaction of PpnN with a substrate analogue, reaction products, and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison to the homologous plant cytokinin-producing LOG proteins reveals that PpnN utilizes an evolutionarily conserved purine hydrolysis mechanism, which in bacteria is regulated by alarmones during stress.

Muscle-specific receptor tyrosine kinase (MuSK) is a single-pass transmembrane protein expressed on skeletal muscle. MuSK is activated by binding of nerve-derived agrin with the help of muscle coreceptor LRP4, leading to the clustering of acetylcholine receptors (AChR), which is required for the formation and maintenance of functional neuromuscular junctions. The structural mechanism of MuSK activation by physiological and artificial agonistic agents has remained elusive. In this study, we isolated a 27-residue linear peptide (L1) that binds human MuSK with high affinity. Genetic fusion of L1 to either the N or C termini of the human IgG Fc resulted in two different versions of MuSK dimerizers, denoted as L1-Fc and Fc-L1. Only Fc-L1 activated MuSK on myotubes and induced AChR clustering. Crystallographic analysis of MuSK-L1 interactions revealed that MuSK activation requires a particular dimeric conformation, pointing toward the importance of the lateral size of the receptor complex at the muscle cell surface.

Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme responsible for propionyl-CoA catabolism. Deficiencies in human PCC (hPCC) cause propionic acidemia, a severe metabolic disorder driven by toxic metabolite accumulation. Despite its therapeutic relevance, the structural basis of hPCC's catalytic function remains unresolved. Here, we present high-resolution cryo-EM structures of hPCC in four distinct states, unliganded, ADP-, AMPPNP-, and ATP-bound/substrate-bound, capturing the full trajectory of the biotin carboxyl carrier protein (BCCP) domain as it translocates between active sites. Our results reinforce the crucial role of nucleotide-gated B-lid subdomain in synchronizing catalysis through coupling with BCCP movement. Structural and biochemical analysis of 10 disease-associated variants reveals how mutations disrupt key domain interfaces and dynamic motions required for activity. These new insights define the mechanistic principles governing hPCC functions, establish a structural framework for understanding PCC-related disorders, and lay the groundwork for future efforts to engineer functional replacements or modulators for metabolic therapy.

Ca-triggered exocytosis from neurons and endocrine cells is regulated by neuronal soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Conformational changes in syntaxin-1-the plasma membrane t-SNARE-are essential for vesicle docking and exocytosis. The nature of these conformational changes on the plasma membrane in living cells, however, remains largely unknown. Here, we develop a fluorescence system to map short-range conformational changes in syntaxin-1a in native plasma membranes of unroofed cells. We use a fluorescence resonance energy transfer (FRET) technique that employs site-specific protein labeling with unnatural fluorescent amino acids as donor fluorophores and colored transition metal ion acceptors bound to engineered di-histidine sites to map angstrom-scale distances. We find that phosphatidylinositol 4,5-bisphosphate (PIP2) regulates a conformational change in syntaxin-1a by modulating the structure of syntaxin-1a and its interaction with Munc18-1. Our results uncover new regulatory mechanisms of syntaxin-1a by PIP2 in the steps leading to Ca-triggered exocytosis.