Journal Of Structural Biology[JOURNAL]
Sun
200 papers
Crystal structure of Escherichia coli type I signal peptidase P84A in complex with lipopeptide antibiotic arylomycin A.
Luo C, Paetzel M
J Struct Biol · 2025 Dec · PMID 41207487 · Publisher ↗
Type I signal peptidase (SPase I) is an essential membrane-bound enzyme that removes amino-terminal signal peptides from secretory proteins. Owing to its critical role in bacterial viability and its periplasmic accessibi...
Type I signal peptidase (SPase I) is an essential membrane-bound enzyme that removes amino-terminal signal peptides from secretory proteins. Owing to its critical role in bacterial viability and its periplasmic accessibility, SPase I has emerged as an attractive target for antibiotic development. Arylomycins, a class of macrocyclic lipohexapeptide natural products, inhibit SPase I by binding to its active site. Previous studies have identified a key resistance determinant-a proline residue at the base of the substrate-binding groove (Pro84 inEscherichia coliSPase I)-which reduces arylomycin affinity. Here, we present the crystal structure of theE. coliSPase I P84A mutant in complex with arylomycin A, revealing that the introduced alanine enables an additional hydrogen bond between the enzyme backbone and the arylomycin N-terminal carbonyl, thus enhancing the affinity for arylomycins. Furthermore, a newly developed preprotein-binding assay utilizing a non-cleavable version of ProOmpA Nuclease A demonstrates that substituting SPase I Pro84 with serine or leucine disrupts substrate recognition, underscoring the delicate balance between inhibitor resistance and substrate processing. These findings reveal that residue Pro84 participates in the interaction between preprotein signal peptides and the E. coli SPase I substrate-binding groove, offering a foundation for designing next-generation arylomycin analogs with improved antibacterial potency.
Cholesterol crystals in reservosomes of Trypanosoma cruzi.
Estevam H, Carvalho RT, Salgado LT … +5 more , Alcantara CL, Aguiar-Seabra J, de Souza W, Cunha-E-Silva NL, Pereira MG
J Struct Biol · 2025 Dec · PMID 41176037 · Publisher ↗
The LDL endocytosis provides cholesterol supply to Trypanosoma cruzi epimastigotes. Cholesterol reaches reservosomes (lysosome like organelles) being used according to cell demand or is storage in lipid droplets. But a r...
The LDL endocytosis provides cholesterol supply to Trypanosoma cruzi epimastigotes. Cholesterol reaches reservosomes (lysosome like organelles) being used according to cell demand or is storage in lipid droplets. But a remnant fraction remains in reservosome lumen where solidifies. In this work we investigated the crystalline properties of these cholesterol solids. First, ultrathin sections, freeze fracture and deep etching replicas suggested collectively different spatial configurations such as needles, plaques or rounded structures. Cryo-EM images showed hemi- and membrane profiles in close association with sterol solids, possibly flanking the growth of these structures. Second, the analysis in situ of parasites by polarized light microscopy pointed to the birefringence of cholesterol. In this way, we used fractions of reservosome lipid inclusions to determine the spectral signature by FTIR, and X-ray diffraction defined the crystallinity of the lipid inclusions. Additionally, our analyses showed that cholesterol was arranged in two polymorphs of anhydrous crystal. Cholesterol crystals had triclinic configuration. Polymorph 1 presented the following unit cell parameters: a = 14.21Å, b = 33.86Å, c = 10.56Å, V = 5028.8Å while the polymorph 2: a = 27.32 Å, b = 38.24 Å, c = 10.66 Å, V = 9776.98 Å. Differences in crystalline densities were also found by our group. The polymorph 1 was more packed and denser than the second crystal analyzed. The densities were estimated in 5.11 g/cm and 2.63 g/cm, respectively. Third, cholesterol crystals did not impair metacyclogenesis being rapidly dismantled if parasites were kept under nutritional starvation.
Membrane and vesicle structure detection in cryo-electron tomography based on deep learning.
Morales-Martínez A, Garduño E, Carazo JM … +2 more , Sorzano COS, Vilas JL
J Struct Biol · 2025 Dec · PMID 41176036 · Publisher ↗
Cryo-electron tomography (cryo-ET) is a microscopy technique that enables the acquisition of 3D images of biological samples. Research in cell biology has shown that cellular processes are carried out by groups of macrom...
Cryo-electron tomography (cryo-ET) is a microscopy technique that enables the acquisition of 3D images of biological samples. Research in cell biology has shown that cellular processes are carried out by groups of macromolecules that interact in a crowded environment. In such an environment, where multiple biological macromolecules coexist and intertwine, semantic segmentation becomes even more challenging but crucial to understanding the structure and function of macromolecular complexes. However, manual semantic segmentation can be time-consuming, highly subjective, and prone to variability, which poses significant obstacles in studies dealing with large volumes of data. In contrast, automated algorithms such as Convolutional Neural Networks (CNNs) can process large-scale datasets with minimal human resources, thereby reducing the subjectivity associated with manual segmentation. In this work, we propose a convolutional neural network architecture that combines the features of U-Net, DeepLab, SegNet, Gated-SCNN, LSTM (Long Short-Term Memory), RNN (Recurrent Neural Network), and GAN (Generative Adversarial Network) architectures. This hybrid architecture effectively learns to identify different types of membranes and can replicate the behavior of a skilled human annotator. This system demonstrates a strong ability to segment various cellular membranes and vesicle structures.
Deep geometric framework to predict antibody-antigen binding affinity.
Bandara N, Premathilaka D, Chandanayake S … +5 more , Hettiarachchi S, Varenthirarajah V, Munasinghe A, Madhawa K, Charles S
J Struct Biol · 2025 Dec · PMID 41138893 · Publisher ↗
In drug development, the efficacy of an antibody depends on how the antibody interacts with the target antigen. The strength of these interactions, measured through "binding affinity", gives an indication of how successf...
In drug development, the efficacy of an antibody depends on how the antibody interacts with the target antigen. The strength of these interactions, measured through "binding affinity", gives an indication of how successful an antibody is in neutralizing an antigen. Due to the high computational complexity of traditional techniques for binding affinity quantification, deep learning is recently employed for the task at hand. Despite the commendable improvements in deep learning-based binding affinity prediction, such approaches are highly dependent on the quality of the antibody-antigen structures and they tend to overlook the importance of capturing the evolutionary details of proteins upon mutation. Further, most of the existing datasets for the task only include antibody-antigen pairs related to one antigen variant and, thus, are not suitable for developing comprehensive data-driven approaches. To circumvent the said complexities, we first curate the largest and most generalized (i.e., including a wide array of antigen variants) datasets for antibody-antigen binding affinity prediction, consisting of more than 100K sequence pairs, 8K structure pairs and the corresponding continuous binding affinity values. Subsequently, we propose a novel deep geometric neural network comprising a structure-based model, which is to account atomistic-scale structural features, and a sequence-based model, which is to attribute sequential and evolutionary information, while sharing the learned information from each model through cross-attention blocks. Further, within each parallel model, we mimic the interaction space of antibodies and antigens through a set of multi-scale hierarchical attention blocks and the final latent vectors of each model are obtained by considering antibody and antigen representative vectors and the interaction vector. The proposed framework exhibited a 10% improvement in mean absolute error compared to the state-of-the-art models while showing a strong correlation (>0.87) between the predictions and target values. Additionally, we extensively discuss the model optimization strategies, weight space analysis, and interpretability in a post-hoc fashion. We release our datasets and code publicly to support the development of antibody-antigen binding affinity prediction frameworks for the benefit of science and society.
Helicon: Helical parameter determination and 3D reconstruction from one image.
Li D, Zhang X, Jiang W
J Struct Biol · 2025 Dec · PMID 41130582 · Full text
Helical symmetry is a common structural feature of many biological macromolecules. However, determination of the helical parameters and de novo 3D reconstruction remain challenging. We have developed a computational meth...
Helical symmetry is a common structural feature of many biological macromolecules. However, determination of the helical parameters and de novo 3D reconstruction remain challenging. We have developed a computational method, Helicon, which poses helical reconstruction as a linear regression problem with the projection matrix parameterized by the helical twist, rise, and axial symmetry. A sparse search of the twist and rise parameters would allow determination of helical parameters and 3D reconstruction directly from one 2D class average or a raw cryo-EM image. The Helicon method has been validated with simulation tests and experimental cryo-EM images of helical tubes, non-amyloid filaments, and amyloid fibrils. Imaging stitching and L1 regularization of linear regression were shown to improve the robustness for low-twist amyloids and noisy raw cryo-EM images. Using Helicon, we could successfully determine the helical parameters and perform de novo reconstruction of a previously unreported, low-abundance tau amyloid structure from a publicly available dataset.
Local microenvironments of capsomer variants in the PBCV-1.
Guo W, Alarcon E, Sanchez JE … +2 more , Xiao C, Li L
J Struct Biol · 2025 Dec · PMID 41125214 · Full text
PBCV-1, a giant virus classified among the Nucleocytoviricota virus (NCV) whose structure has been determined to near atomic resolution. The majority capsomers forming the capsid of PBCV-1 are Type I capsomers while five...
PBCV-1, a giant virus classified among the Nucleocytoviricota virus (NCV) whose structure has been determined to near atomic resolution. The majority capsomers forming the capsid of PBCV-1 are Type I capsomers while five other type of variants have been found in recent high resolution structure. Interestingly, some variants, such as Type V capsomers, are found at particular capsid locations whose roles are unclear. To reveal the roles of a Type V capsomer, we replaced the Type V capsomer by a Type I capsomer to compare the interaction among the two types of capsomer variant, especially the interactions between each of the Type V/Type I capsomer and its local capsid microenvironment. Our results revealed significant differences between Type V and Type I capsomers. Notably, the Type V capsomer demonstrated a stronger binding force to the surrounding capsomers than the Type I capsomer. Moreover, the identified salt bridges between Type V/I capsomers and their surrounding capsomers corroborate the results of electrostatic calculations, further highlighting the important residues involved in these interactions. Understanding these local capsid microenvironments will be essential to elucidate the mechanisms governing viral capsid assembly.
Changes in Arabidopsis thaliana seedling cell wall assembly induced by treatment with Yariv reagent - Molecular features & visualization with immunocytochemistry and a fluorescent Yariv reagent.
Leszczuk A, Kutyrieva-Nowak N, Rueda S … +1 more , Basu A
J Struct Biol · 2025 Dec · PMID 41109487 · Publisher ↗
Arabinogalactan proteins (AGPs) are cell wall-plasma membrane proteins with a high level of glycosylation. The selective and high-affinity binding between AGP and the Yariv reagent has been widely used to carry out funct...
Arabinogalactan proteins (AGPs) are cell wall-plasma membrane proteins with a high level of glycosylation. The selective and high-affinity binding between AGP and the Yariv reagent has been widely used to carry out functional studies on AGPs by disrupting AGP functions using a non-genetic tool. The current work aimed to determine the molecular features of cell walls during Arabidopsis thaliana seed germination under conditions where AGP functions are blocked. To achieve this, we used molecular & imaging methods with molecular probes and for the first time - a new tool for AGP detection - a fluorescent analogue of the Yariv reagent. Themost significant changes included a decrease in the content of AGPs, due to the addition of the Yariv reagent, and subsequent changes only in the content of AGPs upon transfer from the Yariv reagent to fresh Yariv-free medium. Additionally, as a result of the presence of the Yariv reagent, changes in the molecular masses of the analysed cell wall components were observed: lack of AGPs with small molecular mass and disappearance of homogalacturonan with high molecular mass. This work provided the first example of AGP labelling using antibodies and AzYariv-Cy5, and highlights the utility of AzYariv-Cy5 as a broad-spectrum tool for AGP studies.
Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM.
Skrajna A, Lenger C, Robinson E … +8 more , Cannon K, Sarsam R, Ouellette RG, Abotsi AM, Brennwald P, McGinty RK, Strauss JD, Baker RW
J Struct Biol · 2025 Dec · PMID 41083086 · Full text
Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air-water interface often leads to denaturation, aggregation, or a preferred or...
Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air-water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air-water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the "resolution revolution" of cryo-EM. Here, we provide a straightforward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found the lipid affinity grids concentrate particles away from the AWI in thin ice (∼30 nm). We determined three structures ranging from 2.4 to 3.0 Å resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 Å structure of a sub-100 kDa protein without symmetry, demonstrating the utility for a range of biological macromolecules. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air-water interface, and thicker ice.
Prediction of a structural change in the orientation of the cytoplasmic signaling unit of human Toll-like receptor 9 upon binding of agonistic and antagonistic DNA molecules.
Hoshi K
J Struct Biol · 2025 Dec · PMID 41077229 · Publisher ↗
Toll-like receptor 9 (TLR9) recognizes pathogenic DNA molecules containing unmethylated cytosine-phosphate-guanine motifs (CpG DNA) and initiates signaling cascades essential for enhancing immune responses. TLR9 is a typ...
Toll-like receptor 9 (TLR9) recognizes pathogenic DNA molecules containing unmethylated cytosine-phosphate-guanine motifs (CpG DNA) and initiates signaling cascades essential for enhancing immune responses. TLR9 is a type I transmembrane receptor comprising an N-terminal leucine-rich repeat (LRR) domain, a transmembrane domain, and a C-terminal Toll/interleukin-1 receptor (TIR) domain. Most studies have focused on the interaction between the LRR domain and its DNA ligands. However, the TIR domain is crucial for interacting with adapter proteins such as myeloid differentiation factor 88 (MyD88). The aim of this study was to predict changes in the orientation of the TIR domain in human TLR9 (hTLR9) and its complexes with agonistic or antagonistic DNA molecules using the AlphaFold server. AlphaFold predicted the overall structure of hTLR9 with high confidence scores, including part of the TIR domain. Interestingly, binding of agonistic and antagonistic DNA molecules to the N-terminal LRR domain induced a structural change in the orientation of the TIR domain compared to the unbound TLR9 structure. The TIR domain in the predicted hTLR9 model displayed a secondary structure similar to that of the previously reported human TLR1 crystal structure. The predicted model suggests that ligand binding to the N-terminal LRR domain causes a change in the orientation of the TIR domain of hTLR9, likely due to bending of the transmembrane region.
Hurdles and advancements in experimental membrane protein structural biology.
Bajaj R
J Struct Biol · 2025 Dec · PMID 41027549 · Publisher ↗
This short review article traces the evolution of membrane protein structural biology over time and describes various challenges faced and overcome by researchers in the field, highlighting some of the major breakthrough...
This short review article traces the evolution of membrane protein structural biology over time and describes various challenges faced and overcome by researchers in the field, highlighting some of the major breakthroughs and advancements in the field. It presents a thematic exploration of membrane protein structural biology emphasizing on persistent technical and conceptual challenges from protein expression to structural techniques shaping the field with landmark innovations advancing our ability to determine membrane protein structures. The review specifically focus on a few key areas: sourcing and expressing membrane proteins, developing purification strategies and membrane mimetics, and the emergence of powerful structural tools such as X-ray crystallography, cryo-electron microscopy (cryo-EM) and micro-electron diffraction (MicroED). Each section discusses major advancements addressing long standing bottlenecks and opening avenues to understand structure-function relationships in membrane proteins. Furthermore, it also briefly discusses the impact of important discoveries and future perspectives for the field. The review concludes by discussing current emerging frontiers in the field including in-situ structural methods, AI driven structure prediction and future directions for integrative and dynamic membrane protein research.
Precise ligand-selective mechanism at the fab domain of a tau-recognizing antibody.
Tsuchida T, Tsuchiya T, Minoura K … +5 more , In Y, Miyamoto K, Taniguchi T, Ishida T, Tomoo K
J Struct Biol · 2025 Dec · PMID 40983115 · Publisher ↗
Insoluble aggregated tau protein in the form of paired helical filaments is a causative agent of the neurofibrillary tangles observed in Alzheimer's disease (AD). The hexapeptide VQIINK located in the microtubule-binding...
Insoluble aggregated tau protein in the form of paired helical filaments is a causative agent of the neurofibrillary tangles observed in Alzheimer's disease (AD). The hexapeptide VQIINK located in the microtubule-binding domain of tau plays a crucial role in the abnormal aggregation process. Therefore, targeting the VQIINK sequence with a tau aggregation inhibitor may be a promising therapeutic approach for AD. A previous study demonstrated that the Fab domain of the tau antibody (Fab2r3) inhibits tau aggregation by binding to the VQIINK sequence. By determining the three-dimensional structures of the Fab2r3-VQIINK peptide complex and apo Fab2r3, we elucidated the recognition mechanism between Fab2r3 and the VQIINK peptide. However, the basis for the selectivity of Fab2r3 for VQIINK was not completely clear. Therefore, the objective of this report is to investigate the selective binding mechanism of Fab2r3 against VQIINK peptide. Through isothermal titration calorimetry, we show that Ile-4 in the VQIINK peptide is crucial for the selectivity of Fab2r3. X-ray structural analysis of three complexes of Fab2r3 with Ile-4 mutated peptides (VQIVYK, VQILNK, and VQIFNK) suggested that the rigid conformation of a hydrophobic pocket in Fab2r3 plays a vital role in ligand selectivity. These findings may explain the effectiveness of Fab2r3 as a tau aggregation inhibitor.
Mind the corner: Fillets in cryo-FIB lamella preparation to minimise sample loss caused by stress concentration and lamella breakage.
Gorelick S, Velamoor S, Cleeve P … +4 more , Trépout S, Ying L, Naranbhai V, Ramm G
J Struct Biol · 2025 Dec · PMID 40983114 · Publisher ↗
Cryo-FIB milling of biological specimens is a critical and limiting step in the cryo-electron tomography workflow. Preparing electron-transparent cryo-lamellae is a serial, low-throughput process. Even with automation, a...
Cryo-FIB milling of biological specimens is a critical and limiting step in the cryo-electron tomography workflow. Preparing electron-transparent cryo-lamellae is a serial, low-throughput process. Even with automation, a skilled operator can typically only produce 15-25 lamellae in a single cryo-FIB session. During sample handling, milling and transfer, the cryo-fixed cells as well as the supporting film layer face various mechanical forces and thermal stresses due to temperature fluctuations. Moreover, after cells are cryo-FIB milled, the resulting thin lamellae continue to endure external forces from mechanical handling and thermal stress. We propose a simple, yet highly effective modification to the standard rectangular milling pattern by implementing "fillets" or corner smoothing providing better mechanical stability. This adjustment helps to avoid sharp corners at the lamella edges, thereby reducing stress concentration. As a result, this modification decreases the likelihood of lamella breakage and improves the overall yield of ready-for-TEM lamellae by over 40 % as verified experimentally.
A Multi-Technique Investigation to Explore the Structural Integrity and Chemical Complexity of the Brachiopod Lingula anatina (Lamarck, 1801) Shells.
Pal PP, Bar S, Bera SK … +2 more , Sahoo D, Ghorai SK
J Struct Biol · 2025 Dec · PMID 40953778 · Publisher ↗
The shell of Lingula anatina, a living representative of early brachiopods, exemplifies a unique organophosphatic biomineralization strategy that integrates mineral phases with organic components for structural enhanceme...
The shell of Lingula anatina, a living representative of early brachiopods, exemplifies a unique organophosphatic biomineralization strategy that integrates mineral phases with organic components for structural enhancement. This study employs scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray diffraction (XRD), and Raman spectroscopy to comprehensively analyse the microstructure, composition, and mineralogy of the shell. SEM imaging reveals distinct regional microarchitectures, from compact fibrous laminae to porous, reticulate layers, indicating functional specialization in structural reinforcement and flexibility. Elemental analyses confirm a calcium-phosphate matrix dominated by fluorapatite and enriched with trace elements like Mg, Mn, and Fe. XRD and Raman data validate the coexistence of crystalline fluorapatite and calcite with significant amorphous phases. These findings highlight Lingula's evolutionary retention of a hierarchical, organic-inorganic composite shell adapted for environmental interaction, structural resilience, and biomineral control.
Structural insights into IMP2 dimerization and RNA binding.
Zorc SA, Munoz-Tello P, O'Leary T … +9 more , Yu X, Karadi Giridhar MN, Hondros AD, Hansel-Harris A, Forli S, Griffin PR, Kojetin DJ, Roy RN, Janiszewska M
J Struct Biol · 2025 Dec · PMID 40946981 · Full text
IGF2BP2 (IMP2) is an RNA-binding protein that contributes to tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; h...
IGF2BP2 (IMP2) is an RNA-binding protein that contributes to tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function.
CRISP: A modular platform for cryo-EM image segmentation and processing with Conditional Random Field.
Chung SC, Chou PC
J Struct Biol · 2025 Dec · PMID 40935164 · Publisher ↗
Distinguishing signal from background in cryogenic electron microscopy (cryo-EM) micrographs is a critical processing step but remains challenging owing to the inherently low signal-to-noise ratio (SNR), contaminants, va...
Distinguishing signal from background in cryogenic electron microscopy (cryo-EM) micrographs is a critical processing step but remains challenging owing to the inherently low signal-to-noise ratio (SNR), contaminants, variable ice thickness, and densely packed particles of heterogeneous sizes. Recent image-segmentation methods provide pixel-level precision and thus offer several advantages over traditional object-detection approaches: segmented-blob mass can be computed to suppress false-positive particles, particle centering can be improved by leveraging the full brightness profile, and irregularly shaped particles can be identified more reliably. However, low SNR makes it difficult to obtain accurate pixel-level annotations for training segmentation models, and, in the absence of systematic evaluation platforms, most segmentation pipelines still rely on ad-hoc design choices. Here, we introduce a modular platform that automatically generates high-quality segmentation maps to serve as reference labels. The platform supports flexible combinations of segmentation architectures, feature extractors, and loss functions, and it integrates novel Conditional Random Fields (CRFs) with class-discriminative features to refine coarse predictions into fine-grained segmentations. On synthetic data, models trained with our reference labels achieve pixel-level accuracy, recall, precision, Intersection-over-Union (IoU), and F scores all exceeding 90%. We further show that the resulting segmentations can be used directly for particle picking, yielding higher-resolution 3D density maps from real experimental datasets; these reconstructions match those curated by human experts and surpass the results of existing particle-picking tools. To facilitate further research, we release our methods as the open-source package CRISP, available at https://github.com/phonchi/CryoParticleSegment.
Characterization of NMDA receptor Allostery modulation.
Liu Y, Song W, Zhong R … +4 more , Zhang J, Wu X, Jia Y, Kou Z
J Struct Biol · 2025 Sep · PMID 40819686 · Publisher ↗
NMDA receptors are subject to numerous endogenous and exogenous allosteric regulations, which are essential for their complex pathophysiological functions in the brain, and serve as a basis for therapeutic targeting. How...
NMDA receptors are subject to numerous endogenous and exogenous allosteric regulations, which are essential for their complex pathophysiological functions in the brain, and serve as a basis for therapeutic targeting. However, the structural basis of many of these allosteric mechanisms remains unclear. In this study, we first utilized AlphaFold to predict the structural conformations of different NMDA receptor subtypes. Subsequent comparative analyses with experimentally resolved protein structures, coupled with validation using disulfide bond formation, revealed the high precision of these computational predictions. Based on these structures, we systematically investigated the allosteric regulation of NMDA receptors using RoseTTAFold-All-Atom. Our findings elucidated the binding sites of several allosteric modulators across different NMDA receptor subtypes and identified the key amino acids required for binding. These results reveal the structural basis of NMDA receptor allosteric regulation, providing new insights into its physiological and pathological roles, and offering potential avenues for drug development.
Sequence/structural/functional relationships between Ganoderma fungal immunomodulatory proteins (gFIPs) and proteins involved in the modulation of immune response.
Schlosserová N, Perrone GCM, Treml J … +3 more , Sgobba MN, Guerra L, Pierri CL
J Struct Biol · 2025 Sep · PMID 40816616 · Publisher ↗
Fungal Immunomodulatory Proteins from Ganoderma species (gFIPs) have garnered significant interest due to their potential therapeutic applications in modulating immune responses. This study investigates the sequence, str...
Fungal Immunomodulatory Proteins from Ganoderma species (gFIPs) have garnered significant interest due to their potential therapeutic applications in modulating immune responses. This study investigates the sequence, structural, and functional relationships of gFIPs with other proteins involved in immune modulation. Utilizing molecular modelling, multiple sequence alignments, and structural superimposition, we analysed two FIP crystallized structures (PDB IDs: 3F3H and 3KCW) alongside homologous sequences from various taxonomic groups. Our results reveal conserved motifs across fungal, bacterial, and human sequences, indicating potential functional similarities. Comparative structural analysis highlights significant conservation in FIP architecture, with variations primarily in the N-terminal regions. Notably, structural alignment with bacterial toxins, such as ADP-ribosylating binary toxin from Clostridium difficile or protective antigen of Anthrax toxin from Bacillus anthracis suggests mechanistic insights into FIP's immunomodulatory actions. Structural similarities between gFIPs and immune-related proteins, such as bacterial toxin-binding domains, antibody fragments, T-cell receptor components, and immune checkpoint regulators (PD-1) suggest their potential involvement in immune response/inflammation signalling pathways. This comprehensive analysis elucidates the structural basis for the diverse biological activities of gFIPs and underscores their potential as therapeutic agents in immune-related diseases.
1-Deoxy-D-xylulose 5-phosphate synthase: structural perspectives on an essential enzyme in isoprenoid biosynthesis.
Gawriljuk VO, Oerlemans R, Reddem ER … +3 more , Gierse RM, Hirsch AKH, Groves MR
J Struct Biol · 2025 Sep · PMID 40784416 · Publisher ↗
Isoprenoids represent one of the largest and functionally diverse class of natural products, playing essential roles in cellular processes across all domains of life. Unlike humans, many pathogenic organisms such as bact...
Isoprenoids represent one of the largest and functionally diverse class of natural products, playing essential roles in cellular processes across all domains of life. Unlike humans, many pathogenic organisms such as bacteria and protozoa produce their isoprenoid precursors through the 2-C-methylerythritol phosphate (MEP) pathway. 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of this pathway. Despite its biological importance and potential as a drug target, structural studies on DXPS were limited due to its intrinsic flexibility and difficulties in crystallisation. Recent advances, including the development of more crystallisation-friendly constructs and the application of single-particle cryo-electron microscopy (cryo-EM), have significantly expanded our structural understanding of DXPS. This review provides a comprehensive overview of the structural insights gained over the past decades, focusing on the overall architecture of DXPS, its catalytic mechanism, and emerging relevance in structure-based drug discovery.
A structural perspective of transmembrane transport of zinc by ZnT and ZIP transporters.
Xie Q, Ding J, Fu C … +3 more , Chen X, Sun Z, Zhou X
J Struct Biol · 2025 Sep · PMID 40706969 · Publisher ↗
Being the second most abundant trace metal in cells, zinc plays critical roles in a variety of cellular activities, serving as a structural or enzymatic co-factor, or a signaling molecule. Dysregulation of cellular zinc...
Being the second most abundant trace metal in cells, zinc plays critical roles in a variety of cellular activities, serving as a structural or enzymatic co-factor, or a signaling molecule. Dysregulation of cellular zinc has been implicated in many pathophysiological conditions including cancer, neurodegenerative diseases and immune system disorders. Therefore, the cellular zinc homeostasis is tightly controlled by various transport proteins. Two solute carrier protein families, ZnT and ZIP transporters, mediate zinc efflux and influx, respectively, and are important players in maintaining the cellular zinc homeostasis. Recent structural advancement of ZnTs and ZIPs has gained new insight into the transport mechanism of zinc by these transporters. In this review, we discuss ZnT and ZIP transporters from a structural perspective to understand the transport mechanism of zinc across biological membranes.
Effects of base temperature, immersion medium, and EM grid material on devitrification thresholds in cryogenic optical super-resolution microscopy.
Mojiri S, Dobbs JM, Faul N … +3 more , Burg TP, Mahamid J, Ries J
J Struct Biol · 2025 Sep · PMID 40683427 · Publisher ↗
Cryogenic correlative light and electron microscopy (cryo-CLEM) is an imaging strategy that integrates specific molecular labeling and molecular resolution structural information. However, there is a resolution gap of mo...
Cryogenic correlative light and electron microscopy (cryo-CLEM) is an imaging strategy that integrates specific molecular labeling and molecular resolution structural information. However, there is a resolution gap of more than two orders of magnitude between diffraction-limited fluorescence microscopy and electron microscopy (EM). Single-molecule localization microscopy (SMLM) performed at cryogenic temperatures promises to bridge this resolution gap. Nevertheless, the high excitation laser powers required for SMLM risk the devitrification of frozen biological samples, leading to perturbation of their native-like state. Here, we investigate how base cooling temperature, immersion medium, and EM grid support materials influence sample devitrification. Using finite element simulations and experimental validation, we show that a cryo-immersion medium enhances heat dissipation for carbon supports, while metallic supports in a cold nitrogen gas medium tolerate higher laser intensities due to lower base temperatures. Gold supports illuminated at 640nm exhibit markedly high laser thresholds, similar to silver-coated grids. Additionally, metallic supports maintain efficient heat dissipation in vacuum-based cryostats. Our findings provide quantitative insights that aid in optimization of cryo-SMLM setups for improved cryo-CLEM imaging.
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