Structure

Single-particle cryo-electron microscopy (cryo-EM) plays an increasingly important role in structure-based drug discovery (SBDD) by revealing target protein-ligand interactions at the molecular level, but its application has largely focused on large- and medium-sized protein complexes. Many therapeutically important targets are smaller than 50 kDa and remain highly challenging for cryo-EM analysis because of low signal-to-noise ratios and difficulties in particle alignment. Here, we describe cryoFAST, an integrated workflow that couples ligand-compatible Fab discovery and structure-guided computational maturation with biochemical triage, fiducial-complex assembly, and single-particle cryo-EM. Through phage-display screening and/or AI-guided design, we developed high-affinity Fab binders of approximately 50 kDa that stabilize the target without disrupting ligand-target interactions and then assembled Fab-target complexes for cryo-EM analysis. Using the therapeutically relevant extracellular domain (~20 kDa) of interleukin-4 receptor (IL-4R) as a proof of concept, we determined its cryo-EM structure at 2.74 Angstrom resolution and established a framework for elucidating ligand-binding modes. This workflow provides a practical, efficient, and potentially broadly applicable route to the structural characterization of small protein targets that are otherwise challenging to study. By extending the applicability of cryo-EM to high-value but technically challenging small targets, cryoFAST may open new opportunities for structure-based drug discovery.

Cryogenic electron microscopy (cryo-EM) has enabled rapid progress in structural biology throughout the last decade. However, cryo-EM sample preparation still represents a major bottleneck in high-resolution structure determination. Many proteins and macromolecular complexes interact with the air-water interface during sample preparation, which can cause protein denaturation, complex disassembly, and preferred particle orientation. Development of methods to alleviate these issues is an active field of investigation. However, a universally applicable solution to this problem has not been found so far. Here, we describe the simple and cost-effective usage of synthetic peptides with defined amino acid sequence to protect cryo-EM samples from complex disassembly and reduce preferred orientation. We show benefits during cryo-EM grid preparation of a large E3 ubiquitin ligase termed the anaphase-promoting complex (APC/C), and the small CDK11:cyclin L:SAP30BP complex.

TWIK-related halothane-inhibited potassium channel 1 (THIK1) is a two-pore domain potassium (K2P) channel highly expressed in microglia that contributes to microglial homeostasis and neuroinflammatory responses. Pharmacological inhibition of THIK1 suppresses tonic K+ currents and attenuates NLRP3-dependent IL-1{beta} release, highlighting THIK1 as a potential therapeutic target for neuroinflammatory disorders. However, the structural basis of THIK1 inhibition remains unclear. Here we report cryo-electron microscopy (cryo-EM) structures of full-length human THIK1 in complex with C101248, a commercially available tool compound, and CVN293, a clinical-stage inhibitor that has completed Phase I evaluation. The two inhibitors occupy a similar site within the inner vestibule formed by TM2 and TM4 beneath the selectivity filter. Consistent with their closely related chemical scaffolds and shared binding site, both inhibitors remodel the Y273 inner gate but differ in their effects on the C-terminal gate. Electrophysiological analyses further demonstrate that the C-terminal gate contributes differently to inhibition by the two compounds. Together, our findings show how chemically related inhibitors exert different effects on THIK1 despite engaging a common binding site.

While cryogenic electron microscopy (cryo-EM) has transformed structural biology, sample preparation remains a major determinant of success. Specifically, the air-water interface (AWI) can damage complexes, impose preferred orientation, and reduce particle density. Here, we introduce cryoSHIELDs, a chemically diverse library of peptide additives designed to passivate the AWI and improve cryo-EM grid preparation. Across 24 protein systems spanning GPCRs, ABC and SLC transporters, soluble and Fab-containing complexes, and enzymes, cryoSHIELDs increased the success rate of structure determination. Individual cryoSHIELDs improved particle density, angular sampling, complex stability, and, for routine targets, map quality and resolution. This toolkit enabled the structural characterization of important targets, including the ABCC6 transporter, revealing unique TMD0-mediated stabilization, and DAMGO-bound -opioid receptor-Gi complex using substantially lower protein concentration. CryoSHIELDs provide a simple, tunable, and broadly applicable platform for more reliable cryo-EM in discovery biology and structure-based drug development.

Respiratory complex I is a large redox-driven proton pump and contributes to the proton motive force required for ATP generation in mitochondria and bacteria. It has been extensively studied with structural and biochemical approaches leading to enhanced understanding of its three- dimensional architecture. Despite significant progress in deciphering its catalytic function, the redox-coupled proton pumping mechanism remains unclear and extensively debated. The three membrane-bound antiporter-like subunits ND2, ND4 and ND5 are responsible for pumping protons from the negatively charged side (N-side) of the membrane to the positively charged side (P-side). Emerging evidence supports the role of ND5 subunit in releasing pumped protons to the P-side of the membrane, but the question whether ND2 and ND4 also contribute to proton release remains unanswered. Here, we performed atomistic molecular dynamics (MD) simulations of an intact and a truncated model of complex I in which ND5 and ND4 subunits are removed in-silico. The simulation data reveals a previously unobserved conformational change in the P-side region of ND2 subunit that occurs much more easily in a truncated model of complex I, as also estimated based on free energy calculations. The conformational change, in part triggered by the artificial exposure of ND2 subunit to lipid bilayer, results in a local hydration of the region, similar to what has been observed in recent cryo-EM structure of plant CI:CIII2 supercomplex. We discuss the functional relevance of observed conformational transitions and identify residues that can be manipulated with biochemical approaches to shed light on proton pumping by complex I.

The physiological function of the human choline transporter-like protein CTL1 (SLC44A1) remains elusive despite its implication in key pathophysiological processes. Here we combine cryo-electron microscopy, native mass spectrometry, lipidomics, molecular dynamics simulations and functional assays to characterise the molecular basis of phospholipid recognition by CTL1 and explore its potential molecular role. High-resolution structures of CTL1 in both monomeric and dimeric states reveal a broad amphipathic cavity and an extracellular domain that accommodate multiple phospholipid species, identifying phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidic acid as endogenous CTL1 ligands. Molecular dynamics simulations show spontaneous phospholipid recruitment into these conserved binding sites accompanied by membrane thinning, curvature and local phospholipid reorganisation, with membrane deformation being enhanced upon dimerisation. Functional analyses in mammalian cells and in reconstituted proteoliposomes failed to detect CTL1-mediated choline transport, supporting a function distinct from canonical choline uptake. Together, our findings suggest that CTL1 acts as a phospholipid-binding protein capable of locally deforming the membrane and provide a structural framework linking phospholipid recognition to membrane organisation. These results offer new insights into potential mechanisms underlying the altered phospholipid homeostasis associated with CTL1 dysfunction in neurological diseases and cancer.

DNA polymerases generally interact with the processivity clamp (DnaN/{beta}-clamp) during replication via clamp-binding motifs (CBMs). Since a CBM was not detected in DNA Polymerase I (PolI) from E. coli, it is generally believed that PolI performs its functions in genome replication and repair, independently of the {beta}-clamp. We observe that, while PolI and {beta}-clamp proteins from E. coli do not interact, their homologs from Mycobacterium tuberculosis bind strongly, and the binding site localises to the Klenow fragment of PolI. The cryo-EM structure (4.9 [A]) of the Mtb-PolI-Klenow: Mtb-{beta}-clamp complex, coupled with continuous heterogeneity analysis of the cryo-EM data and binding experiments, showed that the primary CBM in PolI may be defined by the stretch 457QLSLLDD463. The crystal structure (2.9 [A]) of the {beta}-clamp with the CBM peptide revealed specific interactions that stabilise the peptide within the {beta}-clamp's canonical binding site. Phylogenetic analyses across different bacteria showed that the identified CBM is present only in PolI from members of the order Mycobacteriales. Overall, the interaction between PolI with the {beta}-clamp, mediated by a CBM unique to the Mycobacteriales order, highlights the mechanistic distinctiveness of genome replication in Mtb and phylogenetically related organisms, and has important implications for drug discovery.

Small multifunctional viral membrane proteins called viroporins are attractive drug targets but are difficult to structurally characterize in a lipid-bilayer environment. We have successfully reconstituted the full-length Envelope (E) protein from SARS-CoV-2 in a lipid-bilayer, co-assembled with a native host factor that binds to E, the PDZ domain 2 of ZO1. We have leveraged the enhancement afforded by dynamic nuclear polarization NMR to determine that the ensemble-averaged oligomer state of E is a pentamer. Complexing the protein with a host PDZ domain did not impact the oligomer state, but resulted in structuring of the last four residues of E, and immobilization of the PDZ domain near the lipid surface. Combined with secondary structure information derived from the chemical shifts, these data were used to model an E protein pentamer together with the PDZ protein dimer. Inclusion of a relevant host factor, as well as a complex lipid bilayer that mimics conditions in the ER-Golgi intermediate compartment, approaches native conditions, and addresses the ongoing controversy regarding the functional oligomer state in an infected cell.

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

De novo design of protein binders requires a target structure. However, for flexible targets, such as intrinsically disordered proteins, this structure does not exist until the binder has stabilized the interaction. Such targets are therefore difficult for methods that separate structure generation from sequence design. We introduce GuideFlip, which co-designs structure and sequence through guided discrete flow matching: binder residues are assigned progressively while the complex is re-predicted at each step, allowing the evolving interface to affect the design process. GuideFlip reduces the hydrophobic bias of direct AlphaFold optimization and improves in silico success rates over existing approaches. We release a database of binder candidates for 177 human disordered proteins. Experimentally, we obtain de novo binders to the C-terminus of -synuclein and the disordered amino terminus of RBX1 with hit rates of 13.5% and 41.7%, respectively, and we confirm the epitopes of selected binders by NMR and mutagenesis. Applying GuideFlip to flexibility on the binder side, we design a nanobody that binds the agonist-bound {beta}1-adrenergic receptor in the active state, but not the receptor in its inactive state, with a 75% hit rate and cryo-EM structure confirming the design. GuideFlip enables protein design where bound structures emerge only upon binding.

Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.

GLP-1R is among the most heavily pursued targets in metabolic disease, but its natural agonist is a 30-residue peptide that must be injected. Two oral non-peptide agonists have reached late-stage trials. Cryo-EM studies have already shown that danuglipron and CHU-128, a close analogue of orforglipron, occupy the receptor differently, but not how either interface behaves over time. We ran 500 ns of unbiased all-atom molecular dynamics on GLP-1R bound to GLP-1, danuglipron (PF-06882961) and orforglipron (LY3502970), one trajectory each, in a POPC bilayer with the G protein removed. Although CHU-128 has been simulated before, its analogue, orforglipron, has not. Scored against contacts computed from the deposited structures, under a force field that shares no parameters with previous oral agonist studies, we recovered 80%, 86% and 79% above 50% occupancy. Indexing contact by chemical group, rather than by receptor residue, and normalising by molecular size, separates the two oral compounds: orforglipron carries ring systems dedicated almost entirely to one receptor domain, one of them directing 99.7% of its contact to the extracellular domain, whereas every group of danuglipron engages both. Danuglipron is correspondingly the outlier on measures that do not depend on a chosen threshold: only 33% of its contacts exceed 90% occupancy against 72% and 84%, and it is the only system whose occupancies remain unconverged at 100 ns. With single trajectories, these are observations rather than tested claims, but two drugs of the same class and comparable size occupy this receptor in structurally distinct ways.

Coronavirus spike proteins mediate receptor binding and fusion and are key determinants of viral host range. To bind protein receptors, some CoV spikes undergo conformational changes termed opening, which expose the receptor-binding surface. Yet, the prerequisites and mechanism of spike opening remain poorly understood, especially outside of beta-CoVs, and many CoV spikes have been observed only in the closed conformation. The receptor specificities of gamma-CoVs, for which birds are natural hosts, await in-depth characterisation and their spikes have not been observed to open. Here, we report structures of the spike from an unusual mammalian gamma-CoV isolated from a beluga whale (BwCoV), in closed and open conformations, the latter with two S1B domains erect. BwCoV spike opening is associated with changes throughout S1 that resemble those observed in beta-CoV spikes and similarly propagate into destabilisation of the fusogenic S2 core. Furthermore, the BwCoV-S structure contains an additional density likely corresponding to a fatty acid, consistent with lipid binding stabilising the closed conformation through a pocket distinct from that identified for beta-CoVs. These structural insights reveal conserved principles regulating spike dynamics across coronavirus genera and support lipid binding as a potential modulatory mechanism of spike opening.

Tau amyloid fibrils adopt disease-specific structures across tauopathies, yet the sequence determinants that favor one pathological fold over another remain poorly defined. Here we combine computational saturation mutagenesis and energetic profiling, in vitro aggregation, cryo-EM and cellular seeding assays to identify sequence changes that alter tau fold compatibility. Across six tauopathy fibril structures, approximately 12,000 substitutions revealed that mutational sensitivity is concentrated within interaction networks formed by amyloidogenic motifs rather than uniformly distributed across the fibril core. Comparison of Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE) folds identified residues within the 335-363 region that differentially influence fold compatibility. The S341L/S352I double mutant accelerated aggregation and remodeled packing within the PAM4-containing amyloidogenic region, producing an alternative amyloid architecture in a minimal tau fragment. S341L/S352I biosensors preferentially responded to CTE-derived over Alzheimer's disease-derived seeds, while aggregates generated from recombinant S341L/S352I tau exhibited CTE-like cellular templating behavior. Although these assemblies are not established to reproduce the atomic CTE fold, the results demonstrate that targeted sequence changes can bias tau aggregation and propagation toward a CTE-compatible state. This framework provides a strategy for defining sequence-structure relationships in polymorphic amyloids and developing conformation-selective models and biosensors.

The targeting function of nuclear localization signals (NLSs) is generally considered independent of a protein's native sequence or fold and is readily transferable to heterologous cargos. Contrary to this paradigm, rapid nuclear translocation of phosphorylated STAT1 (pSTAT1) following cytokine stimulation requires importin {beta}, Ran-GTP, and the importin 5 isoform, which recognizes a non-transferable NLS. Here, we present cryo-EM structures of pSTAT1 bound to importin 5, revealing an asymmetric 2:1 complex that diverges from canonical NLS-mediated cargo recognition. Importin 5 occupies the DNA-binding groove of the pSTAT1 dimer, with a single STAT1 N-terminal domain positioning the C-terminal Armadillo repeats 9-10 (S1B domain) orthogonal to the DNA-binding interface. This interface is also targeted by the Ebola virus protein VP24, an antagonist of interferon signaling. We further show that Ran-GTP alone is insufficient to trigger nuclear release of pSTAT1, which additionally requires the exportin CAS. A cryo-EM reconstruction of the CAS-Ran-GTP-5 complex, supported by in vitro competition assays, demonstrates that CAS and pSTAT1 are mutually exclusive ligands for importin 5. Together, these findings define the molecular choreography of cytokine-induced STAT1 nuclear translocation and release, establishing a general paradigm for STAT family signaling.

Atomistic simulations often struggle to capture the full conformational landscape of biomolecular complexes on experimentally relevant timescales, leaving gaps in our understanding of functionally relevant thermodynamics and kinetics. Cryo-electron microscopy (cryo-EM) provides high-resolution structural snapshots spanning multiple conformational states, but translating these static images into dynamic mechanisms remains challenging. Existing approaches for integrating cryo-EM data with molecular simulations either perturb the underlying dynamics through artificial biasing potentials or remain confined to conformations already present in the prior ensemble. We introduce cryoWEight, an automated and extensible Python framework that couples iterative weighted ensemble (WE) simulations with ensemble reweighting against cryo-EM particle images to steer simulation trajectories towards conformational states consistent with cryo-EM populations. We have found that reweighting in the image space introduces a directional bias towards the target distribution even when the prior ensemble has no overlap with the target distribution in the collective variable space. The reweighting requires only the state likelihoods that set the ensemble weights, and any observable that defines these likelihoods can serve as the target distribution, therefore generalizing beyond the application of cryo-EM. cryoWEight is validated on three proteins: chignolin, the N-terminal domain of ribosomal protein L9, and adenylate kinase. The framework converges towards the target conformational states across different sampling strategies and noise levels, providing a scalable route to integrate cryo-EM data with enhanced sampling approaches.

The ST2 receptor is activated by the alarmin cytokine IL-33, inducing and controlling immune responses. ST2-mediated signalling contributes to protection from parasitic infection, but is also involved in asthma, inflammatory bowel disease, fibrosis and allergic inflammation, making inhibitors of ST2 desirable. While signalling is mediated by membrane-associated ST2 on immune cells, extracellular fluids also contain a soluble form of ST2 which acts as a decoy, soaking up IL-33. An effective ST2-targeting therapeutic will selectively target membrane-associated ST2. The intestinal helminth H. polygyrus bakeri suppresses ST2-mediated signalling to reduce helminth clearance from the gut, with the effector HpBARI2 directly binding to and inhibiting ST2. Here, we present the structure of the ST2:HpBARI2 complex. HpBARI2 forms a stable, planar trimeric structure, with three vertices each binding a single ST2. HpBARI2 occludes the IL-33 binding site of ST2 and constraints the receptor in a conformation incompatible with recruitment of signalling co-receptor IL-1RAcP. The trimeric valency of HpBARI2 results in preferential binding to multiple membrane-associated ST2 molecules rather than the soluble decoy receptor. Therefore, HpBARI2 combines steric blockade, conformational restriction and multivalent binding to potently and selectively inhibit ST2 signalling in the context of soluble ST2. This insight will guide design of improved therapeutics which target ST2.

During disease and infestation, pathogens and pests deliver effectors inside their host to manipulate host responses, leading to immune suppression and altered nutrient availability. To be successful, pathogens and pests can produce and secrete large repertoires of effectors, consisting of up to hundreds of proteins. While effector virulence activities are usually studied in isolation, effectors can function together or cooperate to regulate transport and/or activity. An important example is the suppression of effector avirulence activity by plant pathogenic microbe effectors. Moreover, some effectors can physically associate with target host proteins and promote virulence. Here we explored the interaction of a conserved and co-regulated effector pair in aphids. Using a combination of computational modelling and crystallography combined with functional assays, we reveal a novel type of oligomeric assembly of the paired aphid effectors in a hexameric/octameric complex. Our data indicate that different oligomerization states exist among orthologs of this effector complex, suggesting that dynamic dissociation or subunit exchange may occur. Structure-based mutagenesis revealed that complex formation is essential for both effector protein stability and the in planta activity of one of these proteins. These findings emphasize the significance of effector hetero-oligomerization at the plant-insect interface, consistent with the model that this effector pair is an evolutionarily conserved module across aphid species.

Golgins are widely described as long coiled-coil proteins that contribute to the structural organisation and trafficking functions of the Golgi apparatus. Although experimental structures have been determined for a limited number of golgin regions, atomic-level information on their extended coiled-coil segments remains scarce, and the oligomeric state, topology, and register of most predicted regions remain unestablished. Here, we characterise a predicted coiled-coil region of the yeast golgin Bug1 (BUG1cc) using structural, biophysical, and computational approaches. X-ray crystallography revealed a parallel, in-register dimeric coiled-coil containing ten heptad repeats and a predominantly hydrophobic core, with specific polar interactions contributing to dimer stabilisation. In solution, BUG1cc was dimeric under SEC-MALS conditions and remained highly alpha-helical across the pH and ionic strength conditions examined. CD measurements revealed pronounced scan-rate-dependent hysteresis, while DSC independently confirmed an asymmetry between heating and cooling transitions. Increasing protein concentration shifted both apparent transition temperatures while preserving thermal hysteresis, supporting chain association and conformational rearrangements in structural recovery. Structure-based simulations indicated that interface contacts and intra-chain helicity are thermodynamically coupled and melt as a single cooperative unit, and that the monomer released on dissociation is compact and only partially helical, so that reassociation proceeds through a coupled folding-binding mechanism whose rate-limiting step is conformational rather than bimolecular. Together, these results establish the molecular architecture of a predicted coiled-coil region of Bug1 and reveal a complex folding landscape in which oligomerisation, secondary-structure recovery, and kinetic barriers are tightly coupled.

Human {beta}-defensin 1 (hBD-1) is a small, disulfide-constrained antimicrobial peptide produced predominantly in the kidney and urogenital tract, where it contributes to epithelial innate defense. Genetic variation in the gene encoding hBD-1 has been associated with altered expression levels and susceptibility to infections, including infection following solid organ transplantation, yet the molecular consequences of coding substitutions in the active, mature form remain largely undefined. Here, we integrate conventional genomics, protein bioinformatics, structural modeling, and molecular dynamics to investigate the molecular consequences of 93 human-observed hBD-1 missense variants. Evaluation of hBD-1 missense variants across sequence-, structure-, and dynamics-based dimensions reveals variant-specific molecular perturbation signatures that complement conventional and static biophysical predictions. By treating naturally occurring substitutions as a perturbation series across a compact defensin scaffold, this study identifies multiple molecular routes through which sequence variation can remodel an endogenous antimicrobial peptide and prioritizes variants for direct structure-function testing. These findings provide a mechanistic framework for investigating how naturally occurring hBD-1 variation may influence peptide behavior and, ultimately, antimicrobial function.