Journal Of Structural Biology[JOURNAL]
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200 papers
Structural organization of the organic sheath that delineates extracellular seta silicification in diatoms.
Safadi R, Aram L, de Haan D … +2 more , Avrahami EM, Gal A
J Struct Biol · 2025 Jun · PMID 40294667 · Publisher ↗
Nanopatterning of inorganic materials is a challenging task for contemporary science. It is therefore remarkable that unicellular organisms can form intricately shaped biominerals. A prominent example is the silica cell...
Nanopatterning of inorganic materials is a challenging task for contemporary science. It is therefore remarkable that unicellular organisms can form intricately shaped biominerals. A prominent example is the silica cell wall of diatoms, which usually forms in specialized intracellular organelles. Inside such an organelle, biological regulation proceeds via the concerted activity of various organic macromolecules and inorganic precursors. However, it was shown that a specific type of elongated silica structures, called setae, which characterizes the diatom genus Chaetoceros, form extracellularly, raising questions about the regulatory mechanisms of this silicification process. Here, we study a relatively large species, Chaetoceros rostratus, that forms long and intricate setae. We used in-cell cryo electron tomography to image the native state of seta formation. The high-resolution 3D data show that silica formation outside the cell membrane involves continuous organic sheath that covers the newly formed seta. This sheath has an elaborate structure and is positioned tens of nanometers away from the silica by structural macromolecules that might be involved in architectural regulation. Elucidating the structural components of this delicate living system will allow for new opportunities to learn about the biological strategies for controlled mineralization.
Characterization of conformational flexibility in protein structures by applying artificial intelligence to molecular modeling.
Kopylov K, Kirilin E, Voevodin V … +1 more , Švedas V
J Struct Biol · 2025 Jun · PMID 40287075 · Publisher ↗
Recent AI applications have revolutionized the modeling of structurally unresolved protein regions, thereby complementing traditional computational methods. These state-of-the-art techniques can generate numerous candida...
Recent AI applications have revolutionized the modeling of structurally unresolved protein regions, thereby complementing traditional computational methods. These state-of-the-art techniques can generate numerous candidate structures, significantly expanding the scope of structural biology. However, to effectively prioritize these models, a physics-based approach is required to assess the energy landscape. Such integration can bridge the gap between rapid model generation and precise determination of functional conformations. To address this challenge, we propose an integrated approach that combines molecular modeling with AI and HPC. Metadynamics simulations in latent space are used to explore potential energy landscapes based on initial approximations of flexible region structures derived from modeling tools such as AlphaFold, RosettaFold, Modeller, SwissModel, etc. The approach was validated by modeling folding of Trp-cage protein and conformational plasticity of ubiquitin. The predominant conformations of previously unresolved mobile regions in the active center of flavin-dependent 2-hydroxybiphenyl-3-monooxygenase (EC 1.14.13.44) were identified, while estimating the energy associated with these conformational changes.
Determinants of site-selectivity in human ileal bile acid-binding protein by NMR dynamic analysis of a functionally-impaired mutant.
Teski T, Horváth G, Toke O
J Struct Biol · 2025 Jun · PMID 40268173 · Publisher ↗
Human ileal bile acid-binding protein (hI-BABP), a member of the family of intracellular lipid-binding proteins, has a key role in the enterohepatic circulation of bile salts. The two internal binding sites of hI-BABP ex...
Human ileal bile acid-binding protein (hI-BABP), a member of the family of intracellular lipid-binding proteins, has a key role in the enterohepatic circulation of bile salts. The two internal binding sites of hI-BABP exhibit positive cooperativity accompanied by a site preference of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in the human body. Previous study of Q51A hI-BABP in its apo state, a mutant with lost site-selectivity, suggests that disruption of the hydrogen-bonding network in the vicinity of the C/D-turn has long-range dynamic effects. To improve our understanding of the determinants of site-selectivity in hI-BABP, a comparative NMR chemical shift and spin relaxation analysis of homo- and heterotypic bile salt complexes of wild-type and Q51A hI-BABP was carried out. The wild-type GCDA-complex shows a striking similarity with the thermodynamically most stable hI-BABP:GCDA:GCA complex in terms of both structure and dynamic behaviour, suggesting that the bound GCDA at site 1 has a decisive role in conveying key stabilizing interactions in the physiologically most abundant heterotypic complex. Destabilization of hI-BABP-GCDA by the functionally impairing mutation Q51A is indicated by both the increase of ms-timescale motions in key segments of the protein as well as by increased ps-ns local fluctuations superimposed on slow motions. Our study suggests that binding interactions in hI-BABP might be modulated by altering the dynamic behaviour of specific segments in the protein with implications for targeting the intracellular trafficking of bile salts and bile salt-induced stimulation of nuclear receptors.
2.0 Å cryo-EM structure of the 55 kDa nucleoplasmin domain of AtFKBP53.
Bharambe N, Saharan K, Vasudevan D … +1 more , Basak S
J Struct Biol · 2025 Jun · PMID 40262726 · Publisher ↗
The knowledge of three-dimensional structures of biological macromolecules is crucial for understanding the molecular mechanisms underlying disease pathology and for devising drugs targeting specific molecules. Single pa...
The knowledge of three-dimensional structures of biological macromolecules is crucial for understanding the molecular mechanisms underlying disease pathology and for devising drugs targeting specific molecules. Single particle cryo-electron microscopy (Cryo-EM) has become indispensable for this purpose, particularly for large macromolecules and their complexes. However, its effectiveness has been limited in achieving near-atomic resolution for smaller macromolecules. This study presents the Cryo-EM structure of a 55 kDa pentameric AtFKBP53 nucleoplasmin domain at 2.0 Å nominal resolution. Our approach involves selecting the optimal grid for data collection and precise alignment of small particles to enhance the resolution of the final 3D reconstructed map. In this study, we systematically processed cryo-EM dataset of a small molecule to improve alignment, and this data processing strategy can be used as a guidance to process the cryo-EM data of other small molecules.
Phylogenetic analysis and structural studies of heteromeric acetyl-CoA carboxylase from the oleaginous Amazonian microalgae Ankistrodesmus sp.: Insights into the BC and BCCP subunits.
Mavila AM, Vargas JA, Condori E … +11 more , Suclupe Farro EG, Furtado AA, López JM, Gonzalez SL, Pereira HD, Marapara JL, Paredes RR, Cobos M, Castro JC, Garratt RC, Leonardo DA
J Struct Biol · 2025 Jun · PMID 40174731 · Publisher ↗
Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes a...
Acetyl-CoA carboxylase (ACC) is an essential enzyme in fatty acid biosynthesis that catalyzes the formation of malonyl-CoA from acetyl-CoA. While structural studies on ACC components have largely focused on prokaryotes and higher plants, the assembly and molecular adaptations of ACC in microalgae remain underexplored. This study aimed to fill this gap by providing the first structural and evolutionary characterization of both biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) from a microalga (Ankistrodesmus sp.). Phylogenetic analysis revealed distinct evolutionary trajectories for BC and BCCP, with BC forming a chlorophyte-specific clade closely related to other oleaginous species, while BCCP displayed two distinct isoforms within green algae, resulting from gene duplication. The crystallographic structure of BC was solved in its apo (1.75 Å) and ADP-Mg-bound (1.90 Å) states, revealing conserved conformational changes associated with cofactor binding. BCCP from Ankistrodesmus sp. displayed a unique QLGTF/H motif instead of the canonical AMKXM biotinylation motif, suggesting loss of biotinylation capacity. However, the presence of three additional lysines in the protruding thumb loop, with Lys95 as a candidate for biotin attachment, indicates potential compensatory adaptations. SEC-MALS and pull-down assays confirmed the formation of a stable 1:1 BC-BCCP complex, and circular dichroism showed increased thermal stability of the complex, supporting its structural stability. This study highlights unique structural adaptations in Ankistrodesmus sp. ACC, emphasizing the evolutionary plasticity of BC and BCCP. These insights provide a foundation for future investigations into ACC regulation in photosynthetic organisms and offer potential biotechnological applications for optimizing lipid production in microalgae.
Mechanistic insights into cyclodextrins as substrates and inhibitors of GH57 family amylopullulanase from Aquifex aeolicus.
Zhu Z, Li M, Xu Q … +5 more , Huang L, Zhou H, Wang W, Wang Q, Yu F
J Struct Biol · 2025 Jun · PMID 40120836 · Publisher ↗
Maltooligosaccharides (MOs) have gained significant attention in the food and pharmaceutical industries owing to their valuable functional properties, including controlled sweetness, digestibility, and enhanced bioavaila...
Maltooligosaccharides (MOs) have gained significant attention in the food and pharmaceutical industries owing to their valuable functional properties, including controlled sweetness, digestibility, and enhanced bioavailability. However, conventional MOs is production involves complex processing steps and significant production costs. A potential high-efficiency synthesis of specific MOs can be achieved through the ring-opening reaction of cyclodextrins (CDs) catalyzed by amylolytic enzymes. In this study, we analyze the catalytic conversion of α-, β-, and γ-CDs by a GH57 family amylopullulanase from Aquifex aeolicus (AaApu) using thin-layer chromatography (TLC). Our findings demonstrate that AaApu has a substrate specificity for γ-CD, while all three CDs exert competitive inhibition on pullulan hydrolysis. To elucidate the molecular mechanism of CDs as inhibitor and substrate of amylopullulanase, we determined high-resolution crystal structures of AaApu (wild-type and D352N) in complex with α-, β-, and γ-CD through co-crystallization. These findings establish a structure-function framework for understanding the bifunctional nature of CDs as both substrates and inhibitors in GH57 amylopullulanases.
Structural insights into the RNA binding inhibitors of the C-terminal domain of the SARS-CoV-2 nucleocapsid.
Dhaka P, Mahto JK, Singh A … +2 more , Kumar P, Tomar S
J Struct Biol · 2025 Jun · PMID 40113149 · Publisher ↗
The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and tra...
The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and transmission. The C-terminal domain of the N-protein (N-CTD) is essential for encapsidation, contributing to the stabilization of the RNP complex. In a previous study, three inhibitors (ceftriaxone, cefuroxime, and ampicillin) were screened for their potential to disrupt the RNA packaging process by targeting the N-protein. However, the binding efficacy, mechanism of RNA binding inhibition, and molecular insights of binding with N-CTD remain unclear. In this study, we evaluated the binding efficacy of these inhibitors using isothermal titration calorimetry (ITC), revealing the affinity of ceftriaxone (18 ± 1.3 μM), cefuroxime (55 ± 4.2 μM), and ampicillin (28 ± 1.2 μM) with the N-CTD. Further inhibition assay and fluorescence polarisation assay demonstrated RNA binding inhibition, with IC ranging from ∼ 12 to 18 μM and K values between 24 μM to 32 μM for the inhibitors, respectively. Additionally, we also determined the inhibitor-bound complex crystal structures of N-CTD-Ceftriaxone (2.0 Å) and N-CTD-Ampicillin (2.2 Å), along with the structure of apo N-CTD (1.4 Å). These crystal structures revealed previously unobserved interaction sites involving residues K261, K266, R293, Q294, and W301 at the oligomerization interface and the predicted RNA-binding region of N-CTD. These findings provide valuable molecular insights into the inhibition of N-CTD, highlighting its potential as an underexplored but promising target for the development of novel antiviral agents against coronaviruses.
Conformational landscape of the mycobacterial inosine 5'-monophosphate dehydrogenase octamerization interface.
Bulvas O, Knejzlík Z, Filimoněnko A … +2 more , Kouba T, Pichová I
J Struct Biol · 2025 Jun · PMID 40107326 · Publisher ↗
Inosine 5'-monophosphate dehydrogenase (IMPDH), a key enzyme in bacterial purine metabolism, plays an essential role in the biosynthesis of guanine nucleotides and shows promise as a target for antimicrobial drug develop...
Inosine 5'-monophosphate dehydrogenase (IMPDH), a key enzyme in bacterial purine metabolism, plays an essential role in the biosynthesis of guanine nucleotides and shows promise as a target for antimicrobial drug development. Despite its significance, the conformational dynamics and substrate-induced structural changes in bacterial IMPDH remain poorly understood, particularly with respect to its octameric assembly. Using cryo-EM, we present full-length structures of IMPDH from Mycobacterium smegmatis (MsmIMPDH) captured in a reaction intermediate state, revealing conformational changes upon substrate binding. The structures feature resolved flexible loops that coordinate the binding of the substrate, the cofactor, and the K ion. Our structural analysis identifies a novel octamerization interface unique to MsmIMPDH. Additionally, a previously unobserved barrel-like density suggests potential self-interactions within the C-terminal regions, hinting at a regulatory mechanism tied to assembly and function of the enzyme. These data provide insights into substrate-induced conformational dynamics and novel interaction interfaces in MsmIMPDH, potentially informing the development of IMPDH-targeted drugs.
Dengue virus-host interactions: Structural and mechanistic insights for future therapeutic strategies.
Khanra M, Ghosh I, Khatun S … +2 more , Ghosh N, Gayen S
J Struct Biol · 2025 Jun · PMID 40090430 · Publisher ↗
Dengue pathogen, transmitted by mosquitoes, poses a growing threat as it is capable of inflicting severe illness in humans. Around 40% of the global population is currently affected by the virus, resulting in thousands o...
Dengue pathogen, transmitted by mosquitoes, poses a growing threat as it is capable of inflicting severe illness in humans. Around 40% of the global population is currently affected by the virus, resulting in thousands of fatalities each year. The genetic blueprint of the virus comprises 10 proteins. Three proteins serve as structural components: the capsid (C), the precursor of the membrane protein (PrM/M), and the envelope protein (E). The other proteins serve as non-structural (NS) proteins, consisting of NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The virus relies on these NS proteins to expropriate host proteins for its replication. During their intracellular replication, these viruses engage with numerous host components and exploit the cellular machinery for tasks such as entry into various organs, propagation, and transmission. This review explores mainly the relationship between dengue viral protein and host proteins elucidating the development of viral-host interactions. These relationships between the virus and the host give important information on the processes behind viral replication and the etiology of disease, which in turn facilitates the creation of more potent treatment strategies.
NMR and semi-synthesis in synergy to study protein regulation.
Viennet T
J Struct Biol · 2025 Jun · PMID 40089044 · Publisher ↗
Structural biology as a field has advanced immensely in the last few years, but the mechanistic roles of protein disordered regions and their associated post-translational modifications on the molecular level are still p...
Structural biology as a field has advanced immensely in the last few years, but the mechanistic roles of protein disordered regions and their associated post-translational modifications on the molecular level are still poorly understood. Nuclear magnetic resonance offers the possibility to investigate these regions with atomic resolution and understand the effect of protein modification, and thus protein regulation. However, obtaining suitable and well-defined samples is not straightforward. Here, I review some approaches to protein semi-synthesis for nuclear magnetic resonance purposes, and their applications. I hope to demonstrate that these chemical and structural biology techniques create a powerful synergy that enables structural studies of protein regulation.
MRGPRX2 ligandome: Molecular simulations reveal three categories of ligand-receptor interactions.
Maier P, Macht M, Beck S … +5 more , Kolkhir P, Babina M, Kremer AE, Zahn D, Wolf K
J Struct Biol · 2025 Jun · PMID 40086706 · Publisher ↗
INTRODUCTION: Mas-related G protein-coupled receptor (MRGPR) X2 is a mast cell receptor known to be activated by a wide range of ligands of various size, charge and origin. Our aim is to gain a deeper understanding of th...
INTRODUCTION: Mas-related G protein-coupled receptor (MRGPR) X2 is a mast cell receptor known to be activated by a wide range of ligands of various size, charge and origin. Our aim is to gain a deeper understanding of the binding processes of the different MRGPRX2 ligands and the ligand-receptor interactions in order to identify crucial structural elements for receptor activation. MATERIALS AND METHODS: We used the three-dimensional structure of MRGPRX2 described in Nature in 2021 by Cao et al. and Yang et al. to computationally model the interaction between MRGPRX2 and small molecule ligands under simulated physiological conditions. RESULTS: Docking and post-docking samplings of the MRGPRX2 ligandome within the GPCR binding pocket led to the identification of key structural features for protein-ligand interactions. On the ligand side, we obtained an overlay of different molecular patterns or chemical groups by comparing different ligands plotted on the receptor. These key features include at least one protonated amine moiety of MRGPRX2 ligands contributing to one salt-bridge and one π-cation interaction, as well as an extended non-polar domain of the ligand surface that offer hydrophobic segregation and/or π-stacking interactions. In the receptor, we identified amino acids (GLU164, ASP184, PHE101, PHE170, TRP243, PHE244 and PHE257) that specifically interact via hydrogen bonding, salt-bridges, π-cation interactions and π-π stacking with the ligands to direct binding and ultimately receptor activation. DISCUSSION: Our insights into ligand-receptor interaction obtained by molecular modeling can help to predict mast cell activation via MRGPRX2 including adverse reactions, and facilitate the development of MRGPRX2 antagonists for the treatment of mast cell-mediated diseases.
Allosteric inhibition of cytosolic NADP-dependent isocitrate dehydrogenase by oxaloacetate.
Kim DK, Cho HY, Cho HJ … +1 more , Kang BS
J Struct Biol · 2025 Jun · PMID 40064325 · Publisher ↗
NADP-dependent cytosolic isocitrate dehydrogenase (IDH1) plays a crucial role in providing reducing energy in response to oxidative stress through the oxidative decarboxylation of isocitrate. NADPH generated by IDH1 serv...
NADP-dependent cytosolic isocitrate dehydrogenase (IDH1) plays a crucial role in providing reducing energy in response to oxidative stress through the oxidative decarboxylation of isocitrate. NADPH generated by IDH1 serves as an essential cofactor for fatty acid synthesis. The regulation of IDH1 activity is vital for the biological functions of NADPH within cells, and mutations in IDH1 have been implicated in various cancers. In an effort to identify small regulatory molecules for IDH1, we determined the crystal structures of mouse IDH1 complexed with isocitrate and with oxaloacetate. Each IDH1 comprises large and small domains that form an active site, along with a clasp domain that connects two IDH1 molecules for dimerization. Isocitrate was located at the active site in the presence of a magnesium ion, while oxaloacetate was found at a novel site formed by the two clasp domains, in addition to the active site. The activity of IDH1 was diminished in the presence of oxaloacetate and could not be restored by the addition of isocitrate, indicating the presence of allosteric regulation. The activity of the IDH1 H170A mutant, which is unable to bind oxaloacetate in the clasp domain, was unaffected by oxaloacetate. This allosteric regulatory site may serve as a potential target for novel IDH1 inhibitors.
Improving CryoEM maps of symmetry-mismatched macromolecular assemblies: A case study on the flagellar motor.
Singh PK, Iverson TM
J Struct Biol · 2025 Jun · PMID 40054642 · Full text
Advances in cryo-electron microscopy instrumentation and sample preparation have significantly improved the ability to collect quality data for biomolecular structures. However, achieving resolutions consistent with data...
Advances in cryo-electron microscopy instrumentation and sample preparation have significantly improved the ability to collect quality data for biomolecular structures. However, achieving resolutions consistent with data quality remains challenging in structures with symmetry mismatches. As a case study, the bacterial flagellar motor is a large complex essential for bacterial chemotaxis and virulence. This motor contains a smaller membrane-supramembrane ring (MS-ring) and a larger cytoplasmic ring (C-ring). These two features have a 33:34 symmetry mismatch when expressed in E. coli. Because close symmetry mismatches are the most difficult to deconvolute, this makes the flagellar motor an excellent model system to evaluate refinement strategies for symmetry mismatch. We compared the performance of masked refinement, local refinement, and particle subtracted refinement on the same data. We found that particle subtraction prior to refinement was critical for approaching the smaller MS-ring. Additional processing resulted in final resolutions of 3.1 Å for the MS-ring and 3.0 Å for the C-ring, which improves the resolution of the MS-ring by 0.3 Å and the resolution of the C-ring by 1.0 Å as compared to past work. Although particle subtraction is fairly well-established, it is rarely applied to problems of symmetry mismatch, making this case study a valuable demonstration of its utility in this context.
Effects of Gα C-terminal deletion on the intrinsic GDP release/GTPase activity and conformational dynamics.
Kim J, Chung KY
J Struct Biol · 2025 Jun · PMID 40023204 · Publisher ↗
Heterotrimeric G proteins (G proteins) serve as key signaling mediators downstream of G protein-coupled receptors (GPCRs). Comprised of Gα, Gβ, and Gγ subunits, the activation state of Gα, determined by GDP or GTP bindin...
Heterotrimeric G proteins (G proteins) serve as key signaling mediators downstream of G protein-coupled receptors (GPCRs). Comprised of Gα, Gβ, and Gγ subunits, the activation state of Gα, determined by GDP or GTP binding, governs G protein activity. While high-resolution structures of GPCR-G protein complexes have identified the Gα C-terminal 5 residues (i.e., wavy hook) as critical for GPCR binding and coupling selectivity, its influence on Gα's intrinsic biochemical properties remains unclear. Here, we investigated the role of wavy hook truncation in the intrinsic GDP/GTP turnover rate, GTPase activity, and conformational dynamics of Gαs and Gαi1 using BODIPY-labeled nucleotides and hydrogen/deuterium exchange mass spectrometry (HDX-MS). Truncation of the wavy hook significantly altered the GDP/GTP turnover rate, GTPase activity, and conformational flexibility of Gαs, particularly at the p-loop through α1 region, but had minimal impact on Gαi1. These findings reveal subtype-specific effects of the wavy hook on G protein stability and conformational dynamics, highlighting the importance of structural elements in regulating G protein function and their implications for GPCR signaling studies.
Three-dimensional reconstruction of Magnetofaba australis strain IT-1: Magnetosome chain position with respect to flagella.
Monteiro E, Cabral AS, Morillo V … +3 more , Acosta-Avalos D, Lins U, Abreu F
J Struct Biol · 2025 Jun · PMID 40020884 · Publisher ↗
Magnetotactic bacteria (MTB) are a broad and diverse group of Gram-negative prokaryotes that biomineralize magnetosomes, organelles composed of a magnetic nanocrystal of magnetite (FeO) or greigite (FeS) and enveloped by...
Magnetotactic bacteria (MTB) are a broad and diverse group of Gram-negative prokaryotes that biomineralize magnetosomes, organelles composed of a magnetic nanocrystal of magnetite (FeO) or greigite (FeS) and enveloped by a biological membrane. Magnetosomes are arranged in one or more chains intracellularly, which impart a magnetic moment to the cell. These structures permit a passive orientation of the MTB with the geomagnetic field lines (GML), which, when associated with swimming propelled by flagella, originate a phenomenon called magneto-aerotaxis, an important life strategy in a chemical stratified environment. There is a classical model based on elongated cells as vibrios and rods that tries to explain the magneto-aerotaxis. Still, this model raises questions when applied to other morphologies other than elongated cells. Here, we observe the spatial disposition of magnetosomes, motility behavior, and influence of magneto-aerotaxis in Magnetofaba australis strain IT-1, an MTB that achieves high swimming speeds and has some peculiarity in its motility. The three-dimensional reconstruction showed that Mf. australis strain IT-1's magnetosome chain is misaligned with the swimming axis, which makes it impossible to use the classical model to explain magneto-aerotaxis in this MTB. Despite this, Mf. australis strain IT-1 was capable of swimming aligned to the GML. Also, this work studied the influence of the magnetosome and magneto-aerotaxis between populations of Mf. australis strain IT-1 with and without magnetosomes. Our results indicated that the magnetosome presence not only positively influences the movement in Mf.australis strain IT-1 but also can positively impact population growth in these MTB.
Computational Engineering of siderocalin to modulate its binding affinity to the antihypertension drug candesartan.
Vogel K, Moeller J, Bozhanova NG … +4 more , Voehler M, Penk A, Meiler J, Schoeder CT
J Struct Biol · 2025 Mar · PMID 39978741 · Full text
Lipocalin family proteins have been shown to bind a vast array of small molecules and have subsequently been adapted to selectively bind specific ligands. In this study, candesartan, an antihypertension drug, was identif...
Lipocalin family proteins have been shown to bind a vast array of small molecules and have subsequently been adapted to selectively bind specific ligands. In this study, candesartan, an antihypertension drug, was identified to bind mouse and human siderocalin in biomolecular NMR experiments, allowing for structural insights into the candesartan-siderocalin interaction. The ligand binding site was determined through an integrative structural biology approach using in silico ligand docking guided by NMR experiments. Building on this structurally informed binding model, we used rational protein design to modulate the binding pocket for increased or decreased ligand binding affinity. The predicted mutations were evaluated in vitro using isothermal titration calorimetry. This resulted in a mutant with a 50-fold increase in binding affinity in addition to a second mutant with a five-fold decrease in binding affinity. Thus, siderocalins have potential as a scaffold for creation of various ligand binding-based tools, including drug scavengers.
Cryo-iCLEM: Cryo correlative light and electron microscopy with immersion objectives.
Faul N, Chen SY, Lamberz C … +3 more , Bruckner M, Dienemann C, Burg TP
J Struct Biol · 2025 Mar · PMID 39971020 · Publisher ↗
Correlative light and electron microscopy (CLEM) is a powerful tool for investigating cellular structure and function at the molecular level. However, while electron microscopy is often performed to great advantage at cr...
Correlative light and electron microscopy (CLEM) is a powerful tool for investigating cellular structure and function at the molecular level. However, while electron microscopy is often performed to great advantage at cryogenic temperatures, this is not always the case for light microscopy. One key challenge is the lack of cryo-compatible immersion objectives. In recent years, multiple cryoimmersion light microscopy (cryo-iLM) approaches have been described, but these techniques have never been used in correlative approaches. Here we present a novel workflow for correlative cryoimmersion light microscopy and electron cryomicroscopy (cryo-iCLEM). Cryo-electron tomography conducted before and after cryo-iLM reveals that cryo-iCLEM maintains ultra-thin, electron-transparent samples mechanically intact and does not degrade the ultrastructural preservation achieved through plunge-freezing. For cryo-iLM, the sample is first embedded in a viscous immersion medium at cryogenic temperatures and examined with a custom cryo-immersion objective. After cryo-iLM, the immersion medium is dissolved in liquid ethane, allowing for subsequent cryo-EM imaging. We further show that cryo-iCLEM can be used on FIB-lamellae, demonstrating that mechanically sensitive samples remain undamaged. Embedding the sample in the immersion fluid reduces contamination and thus allows data acquisition over many hours. Samples can therefore be examined in detail with the advantage of low bleaching rates of fluorophores at cryogenic temperatures. In the future, we hope that our approach can help improve the performance of many advanced light microscopy techniques when they are applied in the context of cryo-CLEM.
Lack of embryonic skeletal muscle in mice leads to abnormal mineral deposition and growth.
Silva Barreto I, Liebi M, Le Cann S … +7 more , Ahmed S, Nielsen LC, Grünewald TA, Dejea H, Lutz-Bueno V, Nowlan NC, Isaksson H
J Struct Biol · 2025 Mar · PMID 39956301 · Publisher ↗
Developing bones can be severely impaired by a range of disorders where muscular loading is abnormal. Recent work has indicated that the effects of absent skeletal muscle on bones are more severe early in development, wi...
Developing bones can be severely impaired by a range of disorders where muscular loading is abnormal. Recent work has indicated that the effects of absent skeletal muscle on bones are more severe early in development, with rudiment length and mineralization lengths being almost normal in muscle-less limbs just prior to birth. However, the impact of abnormal mechanical loading on the nanoscale structure and composition during prenatal mineralization remains unknown. In this exploratory study, we characterized the mineralization process of humeri from muscle-less limb embryonic mice using a multiscale approach by combining X-ray scattering and fluorescence with infrared and light microscopy to identify potential key aspects of interest for future in-depth investigations. Muscle-less humeri were characterized by initially less mineralized tissue to later catch up with controls, and exhibited continuous growth of mineral particles, which ultimately led to seemingly larger mineral particles than their controls at the end of development. Muscle-less limbs exhibited an abnormal pattern of mineralization, reflected by a more widespread distribution of zinc and homogenous distribution of hydroxyapatite compared to controls, which instead showed trabecular-like structures and zinc localized only to regions of ongoing mineralization. The decrease in collagen content in the hypertrophic zone due to resorption of the cartilage collagen matrix was less distinct in muscle-less limbs compared to controls. Surprisingly, the nanoscale orientation of the mineral particles was unaffected by the lack of skeletal muscle. The identified accelerated progression of ossification in muscle-less limbs at later prenatal stages provides a possible anatomical mechanism underlying their recovery in skeletal development.
Computational identification of B and T-cell epitopes for designing a multi-epitope vaccine against SARS-CoV-2 spike glycoprotein.
Nguyen TL, Nguyen TB, Kim H
J Struct Biol · 2025 Jun · PMID 39947305 · Publisher ↗
Although the peak of the COVID-19 pandemic has passed, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a significant global threat and remains a public health concern. Given the ongoing ris...
Although the peak of the COVID-19 pandemic has passed, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a significant global threat and remains a public health concern. Given the ongoing risk and the substantial loss of life caused by the virus, continuous research into vaccine development is essential. This study employs immunoinformatics approaches to identify T-cell and B-cell epitopes for designing a multi-epitope peptide vaccine candidate targeting the Omicron variant. The proposed vaccine construct comprises 1435 amino acids, including eight linear B lymphocyte, seven cytotoxic T lymphocyte, and five helper T lymphocyte epitopes, along with appropriate adjuvants and linkers. The evaluation of the vaccine revealed high antigenicity, non-allergenicity, non-toxicity, and favorable physicochemical properties. To further assess its efficacy, molecular docking studies were performed to investigate interactions between the vaccine and key immune components, including Toll-like receptors and major histocompatibility complex molecules. Stability of these interactions was confirmed using molecular dynamics simulations in triplicate, conducted over 100 ns using GROMACS 2023 to compute key metrics, such as root mean square deviation, root mean square fluctuation, solvent-accessible surface area, and radius of gyration. The results demonstrate that the multi-epitope vaccine has the potential to elicit strong immune responses against the Omicron variant, providing a promising foundation for further experimental validation and clinical development in COVID-19 vaccine research.
A computational approach to predict the effects of missense mutations on protein amyloidogenicity: A case study in hereditary transthyretin cardiomyopathy.
Pyankov IA, Gonay V, Stepanov YA … +5 more , Shestun P, Kostareva AA, Uspenskaya MV, Petukhov MG, Kajava AV
J Struct Biol · 2025 Mar · PMID 39933599 · Publisher ↗
With many amyloidosis-associated missense mutations still unidentified and early diagnostic methods largely unavailable, there is an urgent need for a reliable computational approach to predict hereditary amyloidoses fro...
With many amyloidosis-associated missense mutations still unidentified and early diagnostic methods largely unavailable, there is an urgent need for a reliable computational approach to predict hereditary amyloidoses from gene sequencing data. Progress has been made in predicting amyloidosis-triggering sequences within intrinsically disordered regions. However, some diseases are caused by mutations in amyloidogenic regions within structured domains that must unfold for amyloid formation. Accurate prediction of amyloidogenic regions requires tools for detecting amyloidogenicity and assessing mutation effects on protein stability. We developed datasets of mutations linked to hereditary ATTR cardiomyopathy and others likely unrelated, evaluating TTR mutants with amyloidogenicity and stability predictors. Notably, the stability predictors consistently indicated that ATTR-related mutations tend to destabilize the TTR structure more than non-ATTR-associated mutations. Using these datasets and newly generated mutation features, we developed a machine learning model SDAM-TTR to predict mutations leading to ATTR cardiomyopathy.
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