Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A'α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins.

Correlating super-resolution fluorescence light microscopy with cryo-electron tomography (SRcryoCLEM) is a feasible way of targeting specific proteins of interest for high-resolution cryo-electron tomography (cryoET) imaging within cells. Among different approaches for performing super-resolution fluorescence microscopy on cryogenically preserved samples, cryo-single molecule localization microscopy (cryoSMLM) offers one of the highest imaging resolutions. Thus far, applications of cryoSMLM in SRcryoCLEM have been limited to targeting a single protein structure at a time, as the available palette of cryo-compatible reversibly photoswitchable fluorescent proteins, required for cryoSMLM imaging, is severely limited. Here, we present rsTagRFP and rsEGFP2 as a compatible pair of red and green fluorescent labels that enables dual-colour cryoSMLM, and thus dual-target SRcryoCLEM, in mammalian cells. We demonstrate the simultaneous targeting and identification of two separate structures, MAP2-decorated microtubules and vimentin intermediate filaments, with 30 nm accuracy and within the same cell.

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.

The structure of the tailed phage is composed of an icosahedral (or elongated icosahedral) head and a spiral symmetrical tail, which are connected by a portal located at a unique vertex of the icosahedron. A series of image-processing methods and tools have been developed to address the asymmetric structures of phages. However, the structural determination in small proteins within the head and flexible proteins of tailed phages remains a significant impediment, further hindering our deep understanding of the structural biology field. In this study, we developed a data-processing strategy for tailed phage and demonstrated its efficacy with three cryo-EM datasets, including podophage T7, siphophage T1, and myophage Mu. The proposed strategy combines conventional icosahedral reconstruction with local refinement and reconstruction and consists of four key modules: icosahedral reconstruction, selection of the unique vertex of the icosahedron, local asymmetric reconstruction and refinement, and local defocus refinement. The strategy has been successfully applied to determine the asymmetric structure of a range of tailed phages, with a particular focus on resolving the small proteins (core proteins and scaffolding proteins) within the head and flexible proteins on the tail. In addition, the local defocus refinement of our strategy approaches the approximate resolution limit of the icosahedral capsid. The proposed strategy is a viable scheme for determining the asymmetric structures of tailed phages, especially in podophages.

Cryogenic electron tomography is an important technique that enables the three-dimensional visualisation of microscopic samples. In cryogenic electron tomography, a series of two-dimensional projection images is acquired from different tilt angles of the sample and computationally reconstructed into a tomogram. The tilt range of the specimen stage is typically limited to a certain angular range. Beyond this range, the sample may become too thick for electrons to penetrate, and mechanical components such as the support grid or holder may obstruct the beam, resulting in a loss of image quality. This angular limitation leads to missing information in the reconstructed tomograms, known as the missing wedge problem. Moreover, the use of low-dose electron imaging and other experimental constraints introduces considerable noise, thereby reducing the signal-to-noise ratio of the reconstructed tomogram. In order to solve the problems of missing wedges and low signal-to-noise ratio of tomograms, the Fillnet tomogram restoration framework was designed in this study. The training pair generation module and the FFT_Unet model are specially designed in this framework to improve the accurate acquisition of three-dimensional features in tomograms. Different loss functions are also designed to improve the model's attention to the special features of the samples.

The RNA binding motif 15 protein (RBM15) binds both RNA and proteins to regulate a wide repertoire of processes in the cell, including the positing of N6 methyladenosine marks on RNA, the silencing of genes on the inactive X-chromosome, and even hematopoiesis. Although its C-terminal SPOC domain has been found to facilitate protein-protein interactions, the structural mechanism that underlies how its three N-terminal RNA recognition motifs (RRMs) interact with RNA remains to be elucidated. In this crowdsourced study, we bioinformatically assessed publicly available, genome-wide RNA 2D structural probing and RNA binding protein (RBP) cross-linking and immunoprecipitation (CLIP) data to identify RNAs that bind with RBM15. Binding assays reveal that the RRMs work in concert to bind stem-loop structured RNA motifs with nanomolar binding affinity. Structural modeling and nuclear magnetic resonance (NMR) spectroscopy analysis suggest that RRMs 2 and 3 are coaxially stacked to form a heterodimer; they create a sandwich-like motif around structured RNA. Altogether, this work provides insight into the structural mechanism by which RBM15 interacts with RNAs to govern biological function.

G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory significance in bacterial gene expression. While extensively studied in eukaryotes, the roles of G4s especially two-tetrad (2G) G4s in prokaryotic systems remain greatly underexplored. In this study, we identified and characterized multiple 2G G4-forming motifs within the hfq gene of Acinetobacter baumannii, a clinically significant and highly resilient pathogen. The RNA chaperone Hfq protein plays a central role in post-transcriptional gene regulation in this organism. Using a combination of in silico prediction and biophysical techniques (NMR, CD spectroscopy, EMSA, fluorescence titration, and ITC), we determined the folding and topology of these motifs into stable G4 structures, particularly in RNA. These G4s showed high-affinity binding with BRACO-19, a known G4 ligand, and preferential interaction with full-length Hfq protein compared to its C-terminally truncated variant, underscoring the role of the glycine-rich C-terminal domain in RNA recognition. Furthermore, BRACO-19-mediated stabilization of these G4 structures resulted in significant downregulation of hfq transcript variants, especially in the glycine-rich region. Collectively, this work uncovers a novel regulatory axis involving G-quadruplexes and Hfq protein in A. baumannii, highlighting G4-Hfq interactions as potential antimicrobial targets and offering a scaffold for the broader exploration of RNA-based regulation in this pathogenic bacterium.

Membranes are essential components of cells and their compartments. They are composed of asymmetric phospholipid bilayers that separate different environments ensuring the physiological functioning of cells. Most phospholipids are synthesized in the endoplasmic reticulum and transported to the target membrane via various routes. Phosphatidic acid is the starting point for all lipid synthesis pathways, following either the Kennedy pathway for phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine or the CDP-DAG pathway for cardiolipin, phosphatidylglycerol and phosphatidylinositol. Many of the enzymes responsible for these synthesis pathways belong to the cytidine diphosphate alcohol phosphotransferase (CDP-AP) family for which a detailed structural and functional understanding is missing. In this review, we focus on the CDP-AP protein family which is divided in two classes, defined by different structures and mechanisms. The CDP-AP members are membrane proteins, and their mode of catalysis follows a bi-bi or ping-pong mechanism. Recent studies on different CDP-AP family members are bringing new molecular insights on these essential proteins. TEASER: CDP-alcohol phosphotransferase proteins are highly diverged in structure while their overall function in phospholipid synthesis is conserved.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.