Guanine-rich oligonucleotide sequences have the potential to form four-stranded structure known as G-quadruplex. These structures are frequently observed in crucial regions of the human genome, including promoter and tel...Guanine-rich oligonucleotide sequences have the potential to form four-stranded structure known as G-quadruplex. These structures are frequently observed in crucial regions of the human genome, including promoter and telomeric regions. Due to their involvement in regulating gene expression and cell division, G-quadruplexes have emerged as promising targets for anticancer drugs. This study investigated interaction of berberine with different forms of DNA within human telomeric region. The results of absorption and fluorescence spectroscopy indicated that conformation of DNA plays an important role in the mode of binding. Circular dichroism suggested that berberine promotes compaction of the unstable quadruplex structure formed under non-saline conditions. Furthermore, interaction of berberine with the stable structures of G-quadruplex resulted in a change in their compactness without altering the type of DNA structure. 3D fluorescence spectra analysis by chemometrics methods showed formation of two distinct species probably attributed to the self-association and specific binding of berberin to the different forms of DNA. It can be also concluded that berberine forms a more stable complex with the human telomeric hybride type G-quadruplex structure compared with the basket type. In conclusion, the findings imply that the successful design of drugs targeting DNA within the human telomere region necessitates careful consideration of the diverse forms of DNA.
Arthropods and especially scorpions often use metal ions to harden their cuticle. In this study we analyse the chitin fibre arrangement, metal content and distribution and mechanical properties of the stingers of two sco...Arthropods and especially scorpions often use metal ions to harden their cuticle. In this study we analyse the chitin fibre arrangement, metal content and distribution and mechanical properties of the stingers of two scorpions: the buthid scorpion Centruroides platnicki (PS) and the diplocentrid scorpion Nebo whitei (NS). Results show that both scorpions incorporate the same elements, Zn, Mn and Ca, but in different locations in the stinger cuticle. While NS uses Zn ions as hardening agent in the epicuticle, PS uses Mn/Ca ions. Interestingly, Zn ions were also found in PS but had no impact on the enhancement of the mechanical properties of the stinger. The use of different metal ions in biological materials is likely to enable precise adjustments of material properties to suit not only mechanical but biological functions as well.
The process of particle picking, a crucial step in cryo-electron microscopy (cryo-EM) image analysis, often encounters challenges due to outliers, leading to inaccuracies in downstream processing. In response to this cha...The process of particle picking, a crucial step in cryo-electron microscopy (cryo-EM) image analysis, often encounters challenges due to outliers, leading to inaccuracies in downstream processing. In response to this challenge, this research introduces an additional automated step to reduce the number of outliers identified by the particle picker. The proposed method enhances both the accuracy and efficiency of particle picking, thereby reducing the overall running time and the necessity for expert intervention in the process. Experimental results demonstrate the effectiveness of the proposed approach in mitigating outlier inclusion and its potential to enhance cryo-EM data analysis pipelines significantly. This work contributes to the ongoing advancement of automated cryo-EM image processing methods, offering novel insights and solutions to challenges in structural biology research.
J Struct Biol
· 2025 Mar · PMID 39880147
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Arrestins halt cell signaling by binding to phosphorylated activated G protein-coupled receptors. Arrestin-1 binds to rhodopsin, arrestin-4 binds to cone opsins, and arrestins-2,3 bind to the rest of GPCRs. In addition,...Arrestins halt cell signaling by binding to phosphorylated activated G protein-coupled receptors. Arrestin-1 binds to rhodopsin, arrestin-4 binds to cone opsins, and arrestins-2,3 bind to the rest of GPCRs. In addition, it has been reported that arrestin-1 is functionally expressed in mouse cone photoreceptors. The structural characterization of arrestins was spearheaded by the elucidation of the crystal structure of bovine arrestin-1. Further progress in arrestin structural biology showed that the general fold of the four vertebrate arrestin subtypes is conserved and that self-association seems to play important physiological roles. In solution, mammalian arrestin-1 has been proposed to exist in a species-dependent equilibrium between monomers, dimers, and tetramers, the activated monomer being the form that binds to photo-activated phosphorylated rhodopsin. However, the nature and function of the oligomers of the different arrestin subtypes are still under debate. This article reviews several structural aspects of arrestin-1 in light of two recent crystal structures of Xenopus arrestin-1, which have provided insights on the structure, self-association, activation, and evolution of arrestins in general, and of arrestin-1 in particular.
Mitochondria are double membrane-bound organelles essential for generating energy in eukaryotic cells. Mitochondria can be readily visualized in 3D using Volume Electron Microscopy (vEM), and accurate image segmentation...Mitochondria are double membrane-bound organelles essential for generating energy in eukaryotic cells. Mitochondria can be readily visualized in 3D using Volume Electron Microscopy (vEM), and accurate image segmentation is vital for quantitative analysis of mitochondrial morphology and function. To address the challenge of segmenting small mitochondrial compartments in vEM images, we propose an automated mitochondrial segmentation method called GCTransNet. This method employs grayscale migration technology to preprocess images, effectively reducing intensity distribution differences across EM images. By utilizing 3D Global Context Vision Transformers (GC-ViT) combined with global context self-attention modules and local self-attention modules, GCTransNet precisely models long-range and short-range spatial interactions. The long-range interactions enable the model to capture the global structural relationships within the mitochondrial segmentation network, while the short-range interactions refine local details and boundaries. In our approach, the encoder of the 3D U-Net network, a classical multi-scale learning architecture that retains high-resolution features through skip connections and combines multi-scale features for precise segmentation, is replaced by a 3D GC-ViT. The GC-ViT leverages shifted window-based self-attention, capturing long-range dependencies and offering improved segmentation accuracy compared to traditional U-Net encoders. In the MitoEM mitochondrial segmentation challenge, GCTransNet achieved state-of-the-art results, demonstrating its superiority in automated mitochondrial segmentation. The code and its documentation are publicly available at https://github.com/GanLab123/GCTransNet.
Despite sharing ∼ 43 % sequence identity and structurally similar individual domains, botulinum neurotoxin (BoNT) serotypes A and E have differences in their properties and domain positioning. BoNT/E has a faster onset o...Despite sharing ∼ 43 % sequence identity and structurally similar individual domains, botulinum neurotoxin (BoNT) serotypes A and E have differences in their properties and domain positioning. BoNT/E has a faster onset of action than BoNT/A. This difference is proposed to be due to conformational differences between BoNT/E and the other BoNT serotypes. Where most serotypes have the light chain (LC) and binding domain (BD) on opposite sides of the translocation domain (TD), BoNT/E forms a more compact shape with direct interactions between residues of the LC and BD. To elucidate the structural basis for the different properties of BoNT/A and BoNT/E, biophysical studies including molecular dynamic (MD) simulations, circular dichroism (CD) and small-angle X-ray scattering (SAXS) were applied to BoNT/A, for comparison against previous work on BoNT/E. MD simulationsat six pH values across the toxin's activation barrier (pH ∼ 5.5), followed by one extra repeat for the pH values below 5.5, revealed a rare event at pH 5 and 5.5 where interactions between a previously identified switch region of BoNT/Aand the BD were lost.This hintedat an increased freedom of movement, thus allowing the region to change from α-helical to a β-hairpin. In good agreement with previous work, CD showed a gradual and small loss of helicity as the pH decreased below pH 5.5, stabilising at pH 4.5. Combined with the relative scarcity of structural changes observed by MD in the switch region required for activity, these results may explain the slower onset of action for BoNT/A compared to BoNT/E.
Transmission electron microscopy, especially at cryogenic temperature, is largely used for studying biological macromolecular complexes. A main difficulty of TEM imaging of biological samples is the weak amplitude contra...Transmission electron microscopy, especially at cryogenic temperature, is largely used for studying biological macromolecular complexes. A main difficulty of TEM imaging of biological samples is the weak amplitude contrasts due to electron diffusion on light elements that compose biological organisms. Achieving high-resolution reconstructions implies therefore the acquisition of a huge number of TEM micrographs followed by a time-consuming image analysis. This TEM constraint could be overcome by extracting the phase shift of the electron beam having interacted with a "low contrast" sample. This can be achieved by off-axis electron holography, an electron interferometric technique used in material science, but rarely in biology due to lack of sensitivity. Here, we took advantage of recent technological advances on a dedicated 300 keV TEM to re-evaluate the performance of off-axis holography on unstained T4 and T5 bacteriophages at room temperature and in cryogenic conditions. Our results demonstrate an improvement in contrast and signal-to-noise ratio at both temperatures compared to bright field TEM images, with some limitations in spatial resolution. In addition, we show that the electron beam phase shift gives information on charge variations, paving the way to electrostatic potential studies of biological objects at the nanometer scale.
Cryptosporidium has gained much attention as a major cause of diarrhea worldwide. Here, we present the first structure of H-2K complexed with a decapeptide from Cryptosporidium parvum Gp40/15 protein (Gp40/15-VTF10). In...Cryptosporidium has gained much attention as a major cause of diarrhea worldwide. Here, we present the first structure of H-2K complexed with a decapeptide from Cryptosporidium parvum Gp40/15 protein (Gp40/15-VTF10). In contrast to all published structures, the aromatic residue P3-Phe of Gp40/15-VTF10 is anchored in pocket C rather than the canonical Y/F at P5 or P6 reported for octapeptides and nonapeptides. The results of in vitro refolding assays and circular dichroism experiments showed that the side chains of P3 and P5 play key roles in Gp40/15-VTF10 peptide binding. However, functional analysis of decapeptide epitopes revealed that the Gp40/15-VTF10 peptide did not elicit a strong CD8T immune response, whereas the decapeptide epitope MEDLE2-INF10 induced a significant CD8 T-cell response in peptide-immunized C57BL/6 mice. Using a model structure of H-2K-INF10 complex, we found that the antigenic decapeptide INF10 exhibits a completely different conformation, with the aromatic anchors P3F and P7F docked into the D and C pockets, respectively, while similar peptide conformation and hydrogen bond interactions between the peptide and major histocompatibility complex were found in the resolved H-2K-SVF9 complex. As the H-2K molecule predominantly prefers octapeptides with a strong anchor of P5 Y/F (or P6 Y/F for nonapeptides) binding to the C pocket, we propose that P7 Y/F in the C pocket may represent a novel binding mode for decapeptides. The results should increase the accuracy of T-cell epitope prediction and support the development of T-cell epitope vaccines against cryptosporidiosis.
Antonino JD, Chaudhary S, Lubberts M
… +8 more, McConkey BJ, Valença CAS, de Aragão Batista MV, Severino P, da Costa Mendonça M, Souto EB, Dolabella SS, Jain S
Cry proteins, commonly found in Gram-positive soil bacteria, are used worldwide as aerial sprays or in transgenic plants for controlling crop pest populations and insect vectors. Via PCR analysis, a spore producing soil...Cry proteins, commonly found in Gram-positive soil bacteria, are used worldwide as aerial sprays or in transgenic plants for controlling crop pest populations and insect vectors. Via PCR analysis, a spore producing soil isolate (BV5) was speculated to encode a Cry gene. Partial nucleotide sequence of the amplified PCR fragment showed homology with the Cry8 genes present in GenBank. A full-length Cry gene was cloned, and the predicted protein sequence grouped the newly isolated Cry protein with other Cry8A present in GenBank with a high possibility of it being a new Cry8. SDS-PAGE and MALDI-TOF mass spectrometry confirmed the expression of a single 135 KDa protein matching uniquely to the putative protein sequence of the BV5 Cry gene. However, bioassay against the coleopteran Anthonomus grandis (Coleopterans are a known Cry8A target), showed no activity. Phylogenetic analysis and homology modelling was performed to characterize the protein structure and function. These analyses suggest a series of mutations in one of the variable loops on the surface of the protein.
Tran MH, Martina CE, Moretti R
… +3 more, Nagel M, Schey KL, Meiler J
J Struct Biol
· 2025 Mar · PMID 39765317
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High-throughput characterization of antibody-antigen complexes at the atomic level is critical for understanding antibody function and enabling therapeutic development. Hydrogen-deuterium exchange mass spectrometry (HDX-...High-throughput characterization of antibody-antigen complexes at the atomic level is critical for understanding antibody function and enabling therapeutic development. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) enables rapid epitope mapping, but its data are too sparse for independent structure determination. In this study, we introduce RosettaHDX, a hybrid method that combines computational docking with differential HDX-MS data to enhance the accuracy of antibody-antigen complex models beyond what either method can achieve individually. By incorporating HDX data as both distance restraints and a scoring term in the RosettaDock algorithm, RosettaHDX successfully generated near-native models (interface root-mean square deviation ≤ 4 Å) for all 9 benchmark complexes examined, averaging 3.6 times more near-native models than Rosetta alone. Near-native models among the top 10 scoring were identified in 3/9 cases, compared to 1/9 with Rosetta alone. Additionally, we developed a predictive metric based on docking results with HDX restraints to identify allosteric peptides in HDX datasets.
The nacre formation process is a fascinating phenomenon involving mineral phase transformations, self-assembly processes, and protein-mineral interactions, resulting in a hierarchical structure that exhibits outstanding...The nacre formation process is a fascinating phenomenon involving mineral phase transformations, self-assembly processes, and protein-mineral interactions, resulting in a hierarchical structure that exhibits outstanding mechanical properties. However, this process is only partially known, and many aspects of nacre structure are not well understood, especially at the molecular scale. To understand the interplay between components-aragonite, protein and chitin-of the structure of nacre observed experimentally, we investigate the interactions of a peptide that is part of the protein lustrin A, identified in the nacreous layer of the shell of the abalone Haliotis rufescens, with the (001) crystal surface of aragonite and the chitin molecule. We report the results of atomistic molecular-modelling calculations and molecular-dynamics simulations of the peptide interacting with both the aragonite surface and the chitin polymer. The peptide shows an energetically favourable binding to the aragonite surface. The interaction of the carboxylic groups of the glutamic unit with the crystalline surface is essential to reproduce the characteristic elastomeric properties of this peptide in nacre.
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids derived from cell membranes that activate the endothelial differentiation gene family of G protein-coupled receptors. Activati...Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids derived from cell membranes that activate the endothelial differentiation gene family of G protein-coupled receptors. Activation of these receptors triggers multiple downstream signaling cascades through G proteins such as Gi/o, Gq/11, and G12/13. Therefore, LPA and S1P mediate several physiological processes, including cytoskeletal dynamics, neurite retraction, cell migration, cell proliferation, and intracellular ion fluxes. The basis for the G-protein coupling selectivity of EDG receptors, however, remains unknown. Here, we present cryo-electron microscopy structures of LPA-activated LPA1 in complexes with G, G, and G heterotrimers Comparison of the three LPA1-G protein structures shows clearly different conformations of intracellular loop 2 (ICL2) and ICL3 that are likely induced by the different Gα protein interfaces. Interestingly, this G-protein interface interaction is a common feature of LPA and S1P receptors. Our findings provide clues to understanding the promiscuity of G-protein coupling in the endothelial differentiation gene family.
Costa-Filho JI, Theveny L, de Sautu M
… +1 more, Kirchhausen T
J Struct Biol
· 2025 Mar · PMID 39710216
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Cryogenic electron tomography (cryo-ET) has rapidly advanced as a high-resolution imaging tool for visualizing subcellular structures in 3D with molecular detail. Direct image inspection remains challenging due to inhere...Cryogenic electron tomography (cryo-ET) has rapidly advanced as a high-resolution imaging tool for visualizing subcellular structures in 3D with molecular detail. Direct image inspection remains challenging due to inherent low signal-to-noise ratios (SNR). We introduce CryoSamba, a self-supervised deep learning-based model designed for denoising cryo-ET images. CryoSamba enhances single consecutive 2D planes in tomograms by averaging motion-compensated nearby planes through deep learning interpolation, effectively mimicking increased exposure. This approach amplifies coherent signals and reduces high-frequency noise, substantially improving tomogram contrast and SNR. CryoSamba operates on 3D volumes without needing pre-recorded images, synthetic data, labels or annotations, noise models, or paired volumes. CryoSamba suppresses high-frequency information less aggressively than do existing cryo-ET denoising methods, while retaining real information, as shown both by visual inspection and by Fourier Shell Correlation (FSC) analysis of icosahedrally symmetric virus particles. Thus, CryoSamba enhances the analytical pipeline for direct 3D tomogram visual interpretation.
Cryo-electron tomography is an imaging technique that allows the study of the three-dimensional structure of a wide range of biological samples, from entire cellular environments to purified specimens. This technique col...Cryo-electron tomography is an imaging technique that allows the study of the three-dimensional structure of a wide range of biological samples, from entire cellular environments to purified specimens. This technique collects a series of images from different views of the specimen by tilting the sample stage in the microscope. Subsequently, this information is combined into a three-dimensional reconstruction. To obtain reliable representations of the specimen of study, it is mandatory to define the acquisition geometry accurately. This is achieved by aligning all tilt images to a standard reference scheme. Errors in this step introduce artifacts into the final reconstructed tomograms, leading to loss of resolution and making them unsuitable for detailed sample analysis. This publication presents algorithms for automatically assessing the alignment quality of the tilt series and their classification based on the residual errors provided by the alignment algorithms. If no alignment information is available, a set of algorithms for calculating the residual vectors focused on fiducial markers is also presented. This software is accessible as part of the Xmipp software package and the Scipion framework.
Oligomers of the SARS-CoV-2 nucleocapsid (N) protein are characterized by pronounced instability resulting in fast degradation. This property likely relates to two contrasting behaviors of the N protein: genome stabiliza...Oligomers of the SARS-CoV-2 nucleocapsid (N) protein are characterized by pronounced instability resulting in fast degradation. This property likely relates to two contrasting behaviors of the N protein: genome stabilization through a compact nucleocapsid during cell evasion and genome release by nucleocapsid disassembling during infection. In vivo, the N protein forms rounded complexes of high molecular mass from its interaction with the viral genome. To study the N protein and understand its instability, we analyzed degradation profiles under different conditions by size-exclusion chromatography and characterized samples by mass spectrometry and cryo-electron microscopy. We identified self-cleavage properties of the N protein based on specific Proprotein convertases activities, with Cl playing a key role in modulating stability and degradation. These findings allowed isolation of a stable oligomeric complex of N, for which we report the 3D structure at ∼6.8 Å resolution. Findings are discussed considering available knowledge about the coronaviruses' infection cycle.
Base-J (β-D-glucopyranosyloxymethyluracil) is an unusual kinetoplastid-specific DNA modification, recognized by base-J containing DNA (J-DNA) binding proteins JBP1 and JBP3. Recognition of J-DNA by both JBP1 and JBP3 tak...Base-J (β-D-glucopyranosyloxymethyluracil) is an unusual kinetoplastid-specific DNA modification, recognized by base-J containing DNA (J-DNA) binding proteins JBP1 and JBP3. Recognition of J-DNA by both JBP1 and JBP3 takes place by a conserved J-DNA binding domain (JDBD). Here we show that JDBD-JBP3 has about 1,000-fold weaker affinity to base-J than JDBD-JBP1 and discriminates between J-DNA and unmodified DNA with a factor ∼5, whereas JDBD-JBP1 discriminates with a factor ∼10,000. Comparison of the crystal structures of JDBD-JBP3 we present here, with that of the previously characterized JDBD-JBP1, shows a flexible α5-helix that lacks a positively charged patch in JBP3. Mutations removing this positive charge in JDBD-JBP1, resulted in decreased binding affinity relative to wild-type JDBD-JBP1, indicating this patch is involved in DNA binding. We suggest that the α5-helix might rearrange upon JBP1 binding to J-DNA stabilizing the complex. This work contributes to our understanding of how JBPs bind to this unique DNA modification, which may contribute to identifying potential drug targets to end the base-J dependent parasite life cycle.
Porphyromonas gingivalis, a key pathogen in periodontal, plays a critical role in systemic pathologiesdiseases by evading host defence mechanisms and invading periodontal tissues. Targeting its virulence mechanisms and o...Porphyromonas gingivalis, a key pathogen in periodontal, plays a critical role in systemic pathologiesdiseases by evading host defence mechanisms and invading periodontal tissues. Targeting its virulence mechanisms and overcoming drug resistance are essential steps toward effective therapeutic development. In this study, we focused on the Carbonic Anhydrase (CA, EC: 4.2.1.1) encoded by P. gingivalis as a potential drug target. We determined the crystal structure of PgiCA γ at a resolution of 2.4 Å and conducted kinetic characterization. The structure revealed that active PgiCA γ forms a trimer, with each monomer comprising a left-handed β-helix capped by a C-terminal α-helix and coordinated to a catalytic zinc ion through three histidine residues. Interestingly, one monomer displayed an atypical α-helix conformation, likely due to close interactions with neighbouring trimers within the crystal lattice (a probable crystallographic artefact). These findings provide new insights into the structural and functional properties of PgiCA γ, emphasizing its potential as a target for the development of novel anti-virulence therapies against P. gingivalis.
Although calcification in the cardiovascular system is highly studied, the mechanisms behind it are not well understood. Current proposed mechanisms focus on cellular processes leading to, or controlling the unwanted min...Although calcification in the cardiovascular system is highly studied, the mechanisms behind it are not well understood. Current proposed mechanisms focus on cellular processes leading to, or controlling the unwanted mineralization in soft tissues. However, extracellular components such as collagen and elastin fundamentally regulate the mechanical properties of heart tissues. Here, we report on a toolkit to control the composition of tissues through the selective digestion of extracellular matrix (ECM) components, which can be used to design disease-specific in vitro models. Using this technique, we show that elastin as well as matrix tissue damage may play major role in cardiovascular calcification. This study highlights a novel approach to understand the role of proteins in soft tissue calcifications and may lead to the development of strategies to treat and prevent these unwanted pathological disorders.
Structure-function relationships are deeply rooted in modern biochemistry and structural biology and have provided the basis for our understanding of how protein structure defines function. While structure-function relat...Structure-function relationships are deeply rooted in modern biochemistry and structural biology and have provided the basis for our understanding of how protein structure defines function. While structure-function relationships continue to provide invaluable qualitative information, they cannot, in principle, provide the quantitative information ultimately needed to fully understand how proteins function and to make quantitative predictions about changes in activity from changes in sequence and structure. These limitations appear to arise from fundamental principles of physics, which dictate that proteins exist as interchanging ensembles of conformations, rather than as static structures that underly conventional structure-function relationships. This perspective discusses the concept of ensemble-function relationships as quantitative relationships that build on and extend traditional structure-function relationships. The concepts of free energy landscapes and conformational ensembles and their application to proteins are reviewed. The perspective summarizes a range of approaches that can provide conformational ensemble information and focuses on X-ray crystallography methods for obtaining experimental protein conformational ensembles. Focusing on enzymes as archetypes of protein function, recent literature examples are reviewed that used ensemble-function relationships to understand how protein residues contribute to function and how changes in protein sequence and structure impact activity, leading to new models and providing previously inaccessible mechanistic insights. Potential applications of conformational ensembles and ensemble-function relationships to protein design are examined. The perspective concludes with current limitations of the ensemble-function relationships and potential paths forward toward the next generation of quantitative ensemble-function models.
Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a la...Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 Å resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed.