Structural Basis Research Articles

Syntactic Parallelism in Muglan

This article explores the syntactic parallelism in Mulan. The data are carried out from the secondary sources. The qualitative and descriptive method is used for analysis. The sixteen sentences are taken from the source and their targets were noted along with the transliteration. The selected sentences were analyzed on the basis of structure, functions and voice. On the basis of syntactic parallelism, the findings have been drawn on the basis of voice, sentence types and sentence function. Most of the Nepali active voices in the source text (ST) have been translated into English active voices in the target text (TT). Therefore, the tendency of active (ST) into active (TT) was very high. Throughout the sampled data, Nepali passive voice (ST) into English passive voice (TT) has very low. Similarly on the basis of sentence types, simple sentences in the ST translated into simple sentences in the TT has higher tendency as compared to compound sentences in the ST translated into compound sentences in the TT. The complex sentences in the ST translated into complex sentences in the TT have highest tendency. Likewise on the basis of sentence function, the declarative sentences in the ST into declarative sentences in the TT has highest tendency. Interrogative sentences in the ST into interrogative sentences in the TT has higher tendency than exclamatory sentences in the ST into exclamatory in the TT.The imperative sentence (ST) into imperative sentence (TT) has lowest tendency.

Structural basis of thiamine transport and drug recognition by SLC19A3

Thiamine (vitamin B1) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane, which is mainly mediated by the two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters lead to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report seven cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enable us to capture different states of SLC19A3’s transport cycle, revealing the molecular details of thiamine recognition and transport. We identify seven previously unknown drug interactions of SLC19A3 and present structures of the transporter in complex with the inhibitors fedratinib, amprolium and hydroxychloroquine. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.

LYCHOS is a human hybrid of a plant-like PIN transporter and a GPCR.

Lysosomes have crucial roles in regulating eukaryotic metabolism and cell growth by acting as signalling platforms to sense and respond to changes in nutrient and energy availability1. LYCHOS (GPR155) is a lysosomal transmembrane protein that functions as a cholesterol sensor, facilitating the cholesterol-dependent activation of the master protein kinase mechanistic target of rapamycin complex 1 (mTORC1)2. However, the structural basis of LYCHOS assembly and activity remains unclear. Here we determine several high-resolution cryo-electron microscopy structures of human LYCHOS, revealing a homodimeric transmembrane assembly of a transporter-like domain fused to a G-protein-coupled receptor (GPCR) domain. The class B2-like GPCR domain is captured in the apo state and packs against the surface of the transporter-like domain, providing an unusual example of a GPCR as a domain in a larger transmembrane assembly. Cholesterol sensing is mediated by a conserved cholesterol-binding motif, positioned between the GPCR and transporter domains. We reveal that the LYCHOS transporter-like domain is an orthologue of the plant PIN-FORMED (PIN) auxin transporter family, and has greater structural similarity to plant auxin transporters than to known human transporters. Activity assays support a model in which the LYCHOS transporter and GPCR domains coordinate to sense cholesterol and regulate mTORC1 activation.

Acne-related UVA-induced facial fluorescence: An exploratory study from physiological properties to tissue structure information

UVA-induced facial fluorescence (UVAF) is recognized as an objective measurement technique to quantify the severity of acne. However, notable inconsistencies in quantitative outcomes have been observed in various studies, possibly due to the fact that different colors of fluorescence represent different pathophysiological implications. This study investigated the pathophysiological importance of UVAF color differences and improved its reliability in assessing acne severity. MIDOO Smart Skin Imager was used to capture UVAF and analyze the correlation between fluorescence colors and acne lesions. Techniques such as two-photon fluorescence microscopy, scanning electron microscopy, Western blot, and High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) were used to examine the biochemical composition and structure of comedonal plugs and follicular casts associated with different fluorescence colors. We found that green fluorescence correlates with non-inflammatory acne lesions (comedones), while orange-red fluorescence shows no correlation with either type of lesion. Green fluorescence is associated with higher levels of keratin, indicating keratinization, while orange-red fluorescence is associated with porphyrin from S. epidermidis. UVAF color differences - orange-red are from porphyrins and green from keratin. This distinction helps to understand the structural and physiological bases of facial fluorescence, with potential implications for clinical evaluations of acne.

The cAMP receptor protein from Gardnerella vaginalis is not regulated by ligands

Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening.

Molecular basis of global promoter sensing and nucleosome capture by the SWR1 chromatin remodeler.

The SWR1 chromatin remodeling complex is recruited to+1 nucleosomes downstream of transcription start sites of eukaryotic promoters, where it exchanges histone H2A for the specialized variant H2A.Z. Here, we use cryoelectron microscopy (cryo-EM) to resolve the structural basis of the SWR1 interaction with free DNA, revealing a distinct open conformation of the Swr1 ATPase that enables sliding from accessible DNA to nucleosomes. A complete structural model of the SWR1-nucleosome complex illustrates critical roles for Swc2 and Swc3 subunits in oriented nucleosome engagement by SWR1. Moreover, an extended DNA-binding α helix within the Swc3 subunit enables sensing of nucleosome linker length and is essential for SWR1-promoter-specific recruitment and activity. The previously unresolved N-SWR1 subcomplex forms a flexible extended structure, enabling multivalent recognition of acetylated histone tails by reader domains to further direct SWR1 toward the+1 nucleosome. Altogether, our findings provide a generalizable mechanism for promoter-specific targeting of chromatin and transcription complexes.

Structural basis for the rescue of hyperexcitable cells by the amyotrophic lateral sclerosis drug Riluzole

Neuronal hyperexcitability is a key element of many neurodegenerative disorders including the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), where it occurs associated with elevated late sodium current (INaL). INaL results from incomplete inactivation of voltage-gated sodium channels (VGSCs) after their opening and shapes physiological membrane excitability. However, dysfunctional increases can cause hyperexcitability-associated diseases. Here we reveal the atypical binding mechanism which explains how the neuroprotective ALS-treatment drug riluzole stabilises VGSCs in their inactivated state to cause the suppression of INaL that leads to reversed cellular overexcitability. Riluzole accumulates in the membrane and enters VGSCs through openings to their membrane-accessible fenestrations. Riluzole binds within these fenestrations to stabilise the inactivated channel state, allowing for the selective allosteric inhibition of INaL without the physical block of Na+ conduction associated with traditional channel pore binding VGSC drugs. We further demonstrate that riluzole can reproduce these effects on a disease variant of the non-neuronal VGSC isoform Nav1.4, where pathologically increased INaL is caused directly by mutation. Overall, we identify a model for VGSC inhibition that produces effects consistent with the inhibitory action of riluzole observed in models of ALS. Our findings will aid future drug design and supports research directed towards riluzole repurposing.

The tree shrew model of Parkinson’s disease: A cost-effective alternative to non-human primate models

The surge in demand for experimental monkeys has led to a rapid increase in their costs. Consequently, there is a growing need for a cost-effective model of Parkinson’s disease (PD) that exhibits all core clinical and pathological phenotypes. Evolutionarily, tree shrews (Tupaia belangeri) are closer to primates in comparison to rodents and could be an ideal species for modeling PD. To develop a tree shrew PD model, we used the 1-methyl-4-phenylpyridinium (MPP+), a metabolite derived from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to induce lesions in dopaminergic neurons of the unilateral substantia nigra. The induced tree shrew model consistently exhibited and maintained all classic clinical manifestations of PD for a 5-month period. The symptoms included bradykinesia, rest tremor, postural instability, and about 50% individuals showed apomorphine-induced rotations, a classic phenotype of unilateral PD models. All these are closely resembled the ones observed in PD monkeys. Meanwhile, this model was also sensitive to L-dopa treatment with a dose dependent manner, which suggested that the motor deficits are dopamine dependent. Immunostaining showed a significant loss of dopaminergic neurons (approximately 95%) in the lesioned substantia nigra, which is a crucial PD pathological marker. Moreover, a control group of nigral saline injection did not show any motor deficits and pathological changes. Cytomorphological analysis revealed that the size of nigral dopaminergic neurons in tree shrews is much bigger than that of rodents and is close to that of macaques. The morphological similarity may be an important structural basis for the manifestation of the highly similar phenotypes between monkey and tree shrew PD models. Collectively, in this study we have successfully developed a PD model in a small animal species that faithfully recapitulated the classic clinical symptoms and key pathological indicators of PD monkeys, providing a novel and low-cost avenue for evaluation of PD treatments and underlying mechanisms.

BRCA2 stabilises RAD51 and DMC1 nucleoprotein filaments through a conserved interaction mode

BRCA2 is essential for DNA repair by homologous recombination in mitosis and meiosis. It interacts with recombinases RAD51 and DMC1 to facilitate the formation of nucleoprotein filaments on resected DNA ends that catalyse recombination-mediated repair. BRCA2’s BRC repeats bind and disrupt RAD51 and DMC1 filaments, whereas its PhePP motifs bind recombinases and stabilise their nucleoprotein filaments. However, the mechanism of filament stabilisation has hitherto remained unknown. Here, we report the crystal structure of a BRCA2-DMC1 complex, revealing how core interaction sites of PhePP motifs bind to recombinases. The interaction mode is conserved for RAD51 and DMC1, which selectively bind to BRCA2’s two distinct PhePP motifs via subtly divergent binding pockets. PhePP motif sequences surrounding their core interaction sites protect nucleoprotein filaments from BRC-mediated disruption. Hence, we report the structural basis of how BRCA2’s PhePP motifs stabilise RAD51 and DMC1 nucleoprotein filaments for their essential roles in mitotic and meiotic recombination.

Structural basis of ligand recognition and activation of the histamine receptor family

Histamine is a biogenic amine that is critical in various physiological and pathophysiological processes, including but not limited to allergic reactions, wakefulness, gastric acid secretion and neurotransmission. Here, we determine 9 cryo-electron microscopy (cryo-EM) structures of the 4 histamine receptors in complex with four different G protein subtypes, with endogenous or synthetic agonists bound. Inside the ligand pocket, we identify key motifs for the recognition of histamine, the distinct binding orientations of histamine and three subpockets that facilitate the design of specific ligands. In addition, we also identify key residues responsible for the selectivity of immethridine. Moreover, we reveal distinct structural features as determinants of Gq vs. Gs or Gs vs. Gi coupling differences among the histamine receptors. Our study provides a structural framework for understanding the ligand recognition and G protein coupling of all 4 histamine receptors, which may facilitate the rational design of ligands targeting these receptors.

Starch-related structural basis and enzymatic mechanism of the different appearances of soft rice

To investigate the fine starch structure characteristics and formation mechanism of high-quality appearance soft rice, two high-quality and low-quality soft rice varieties (HA-SR and LA-SR, respectively) were selected. Differences in appearance quality, fine starch structure, and activity of key enzymes involved in starch synthesis during the grain-filling stage were compared. The results showed that compared with LA-SR, HA-SR were less chalky, more transparent, had larger starch grains, a lower content of shorter chains (DP 6–24), a higher content of longer chains (DP ≥ 25), lower relative crystallinity, fewer ordered structures, more amorphous structures and larger thicknesses of semi-crystalline lamellae. In terms of amylase activity during the grain-filling stage, the AGPase and GBSS activities of HA-SR were higher, and the SBE activity of HA-SR was lower compared to LA-SR. In conclusion, higher AGPase activity can produce a higher filling rate resulting in fuller starch grain in soft rice. Fuller starch grains reduce the chalkiness of soft rice. Higher AGPase and GBSS activities and lower SBE activity can result in soft rice with more long-branched and less short-branched amylopectin. Thus, soft rice has lower relative crystallinity and less ordered structure. These structures may facilitate reduce grain chalkiness and improve grain transparency.

Structural basis for inositol pyrophosphate gating of the phosphate channel XPR1.

Precise regulation of intracellular phosphate (Pi) is critical for cellular function, with XPR1 serving as the sole Pi exporter in humans. The mechanism of Pi efflux, activated by inositol pyrophosphates (PP-IPs), has remained unclear. This study presents cryo-electron microscopy structures of XPR1 in multiple conformations, revealing a transmembrane pathway for Pi export and a dual-binding activation pattern by PP-IPs. A canonical binding site is located at the dimeric interface of SPX domains, and a second site, biased toward PP-IPs, is found between the transmembrane and SPX domains. By integrating structural studies with electrophysiological analyses, we characterize XPR1 as an IPs/PP-IPs-activated phosphate channel. The interplay among its TMDs, SPX domains, and IPs/PP-IPs orchestrates the conformational transition between its closed and open states.

Impact of VP2 structure on antigenicity: comparison of BTV1 and the highly virulent BTV8 serotype.

Bluetongue virus (BTV) is an agriculturally and economically significant insect-borne virus that causes serious illness and death in sheep and other domestic and wild ruminants in large areas of the world. Numerous BTV serotypes exist, and distant serotypes exhibit unique neutralizing antibody profiles, which target the outermost capsid protein VP2. The predominant serotype-specific nature of the antibody response to VP2 is a barrier to the development of broad-spectrum prophylactic BTV vaccine candidates. Although VP2 is the main serotype determinant of BTV, the structural basis of serotype specificity has not been investigated. In this study, we utilized the recently available atomic structure of VP2 with a modeled tip domain to carry out in silico structural comparisons between distant serotypes BTV1 and BTV8. These analyses identified structural differences in the tip domain, positioned at the apex of VP2, and informed the design of mutant VP2 constructs. Dissection of tip domain antigenicity demonstrated that the region of structural difference between BTV1 and highly virulent BTV8 was a target of BTV neutralizing antibodies and that mutation of this region resulted in a loss of neutralizing antibody recognition. This study has for the first time provided insights into the structural differences, which underpin the serotype-specific neutralizing antibody response to BTV.IMPORTANCEThe immune system can protect against virus infection by producing antibodies, which bind and inhibit the virus from infecting the susceptible host. These antibodies are termed neutralizing antibodies and generally target the viral receptor binding protein, such as the VP2 of bluetongue virus (BTV). This pressure from the immune system can drive mutation of the viral protein resulting in escape from antibody-mediated neutralization and the evolution of serotypes, as is the case for BTV. Understanding the structural differences, which underpin the different BTV serotypes, could help guide the design of a BTV vaccine that targets multiple serotypes. In this study, we have mapped the VP2 structural differences between distant serotypes, to a region targeted by neutralizing antibodies, and have demonstrated for the first time how VP2 structure is the fundamental basis of serotype specificity.

Publisher Correction: Structural basis of LRPPRC–SLIRP-dependent translation by the mitoribosome

Publisher Correction: Structural basis of LRPPRC–SLIRP-dependent translation by the mitoribosome

Structural basis of CDNF interaction with the UPR regulator GRP78

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional neurotrophic factor that is a disease-modifying drug candidate for Parkinson’s disease. CDNF has pleiotropic protective effects on stressed cells, but its mechanism of action remains incompletely understood. Here, we use state-of-the-art advanced structural techniques to resolve the structural basis of CDNF interaction with GRP78, the master regulator of the unfolded protein response (UPR) pathway. Subsequent binding studies confirm the obtained structural model of the complex, eventually revealing the interaction site of CDNF and GRP78. Finally, mutating the key residues of CDNF mediating its interaction with GRP78 not only results in impaired binding of CDNF but also abolishes the neuroprotective activity of CDNF-derived peptides in mesencephalic neuron cultures. These results suggest that the molecular interaction with GRP78 mediates the neuroprotective actions of CDNF and provide a structural basis for development of next generation CDNF-based therapeutic compounds against neurodegenerative diseases.

Landscape and soil cover diversity in Polissia and Forest-Steppe of Ukraine

Recent research has revealed the patterns of spatial and temporal variability of crop yields for the Polissia and the Forest-Steppe regions of Ukraine. In addition, the approaches to differentiating agroeconomic and agroecological components in the variability of the productive potential of the territories were developed. Approaches have been developed to quantify the spatial and temporal variability of soil erosion for individual areas of the region. The issue of assessing the impact of erosion processes on crop yields at the regional level remains unresolved. The diversity of landscapes and soil cover in Polissia and the Forest-Steppe of Ukraine was assessed in this article. The research covered 10 administrative regions of the north and northwest of Ukraine, which are located mainly within mixed forests (Polissia) and the eastern European forest steppe (Forest-Steppe). Part of the territory is tangential to the Carpathian mountain forests and the Pontic steppe. Within the study area, 30 WRB soils were identified. The largest area is occupied by Phaeozems Albic (21.7% of the total area of the region). These soils are located mainly in the southern and central parts of the region. In the north of the region, Phaeozems Albic form an almost continuous cover, and to the north, the areas of these soils become island-like. Phaeozems Luvic cover 12.1% of the region's territory. The Phaeozems group also includes Phaeozems Gleyic (0.93%), Phaeozems Haplic (0.13%), and Phaeozems Sodic (1.78%). Albeluvisols Umbric (17.1%) occupy a significant part of the region's territory. These soils occupy the north of the region and extend south along the Dnipro River floodplain. Spatially close to these soils are Albeluvisols Gleyic (12.8%). In the western part of the region, in Prykarpattia, there are Albeluvisols Stagnic (0.82%). The organic carbon content in the soils of the administrative districts of the region in the 0-5 cm layer was 5.2±3.3% and ranged 0.5% to 23.9%. In the layers of 5–15 and 15–30 cm, this figure decreased to 1.9 ± 1.1% and 1.9 ± 1.0%, respectively. In the 30–60 cm layer, the organic carbon content was 1.4 ± 0.8% and ranged 0.1% to 6.1%. The lowest organic carbon content was observed in the north central part of the region, and the highest content was observed in the north-east and north-west of the region. In the south of the region, there was found a strip of soils with moderate organic carbon content. The lowest content of organic carbon is characteristic of Albeluvisols and Podzols, which are located in the north of the region. In turn, the highest content of organic carbon is characteristic of organogenic Histosols, which, together with Albeluvisols and Podzols, form the basis of the soil cover structure of the territories in the northeast and northwest of the region. It should be noted that the agronomic role of these soils is insignificant, and their organic carbon content cannot be considered as a traditional indicator of fertility. Nevertheless, organic carbon is a reliable indicator of the anti-erosion properties of both natural and agricultural soils. The organic carbon content of Chernozems is quite high, which is a factor in the high fertility of these soils and their importance in agricultural production. The organic carbon content of Fluvisols and Gleysols is naturally high. The importance of these hydromorphic soils is due to their high productive potential, which ensures their use in agriculture. It is also worth noting their high seed protection potential. Phaeozems cover a large area of the region and contain a high level of organic carbon. These soils form the basis of agricultural production and forestry in the region. The silt content in the region's soils measured 45.6 ± 8.5%, ranging 19.1% to 57.6%. The north of the region is represented by soils with the lowest silt content (19–41%). In the center, in the west of the region, there is a zone with the highest silt content (53–58%). In other parts of the region, there are transitional levels of silt content in the soil. The clay content in the soils was 30.7 ± 13.8% and ranged 7.4% to 36.0%. The zone with the lowest clay content in soils has been formed in the north. The highest clay content in soils is typical for the southern part in the center of the region. The sand content in soils is highest in the northern part of the region, and the lowest in the southwestern and southern parts of the region.

Dimerization Promotes PKR Activation by Modulating Energetics of αC Helix Conversion between Active and Inactive Conformations.

Protein kinase R (PKR) functions in the eukaryotic innate immune system as a first-line defense against viral infections. PKR binds viral dsRNA, leading to autophosphorylation and activation. In its active state, PKR can phosphorylate its primary substrate, eIF2α, which blocks the initiation of translation in the infected cell. It has been established that PKR activation occurs when the kinase domain dimerizes in a back-to-back configuration. However, the mechanism by which dimerization leads to enzymatic activation is not fully understood. Here, we investigate the structural mechanistic basis and energy landscape for PKR activation, with a focus on the αC helix─a kinase activation and signal integration hub─using all-atom equilibrium and enhanced sampling molecular dynamics simulations. By employing window-exchange umbrella sampling, we compute free-energy profiles of activation, which show that back-to-back dimerization stabilizes a catalytically competent conformation of PKR. Key hydrophobic residues in the homodimer interface contribute to stabilization of the αC helix in an active conformation and the position of its critical glutamate residue. Using linear mutual information analysis, we analyze allosteric communication connecting the protomers' N-lobes and the αC helix dimer interface with the αC helix.

Structural basis of human 20S proteasome biogenesis

New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. Here, to understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observe an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.

Reducing treatment time for robotic radiosurgery by considering path efficiency during node selection.

Robotic radiosurgery treatments allow for precise non-coplanar beam delivery by utilizing a robot equipped with a linac that traverses through a set of predetermined nodes. High quality treatment plans can be produced but treatment times can grow large, with one substantial component being the robot traversal time. The aim of this study is to reduce the treatment time for robotic radiosurgery treatments by introducing algorithms for reducing the robot traversal time. The algorithms are integrated into a commercial treatment planning system. First, an optimization framework for robotic radiosurgery planning is detailed, including a heuristic optimization method for node selection. Second, two methods aimed at reducing the traversal time are introduced. One utilizes a centrality measure focusing on the structure of the node network, while the other is based on the direct computation of traversal times during optimization. A comparison between plans with and without the time-reducing algorithms is made for three brain cases and one liver case with basis in treatment time, plan quality, monitor units, and network structure of the selectednodes. Large decreases in traversal times are obtained by the traversal time reducing algorithms, with reductions of up to 49 % in the brain cases and 31 % in the liver case. The resulting reductions in treatment times are up to 30 % and 13 %, respectively. Small differences in plan quality are observed, with similar dose-volume histograms, dose distributions, and conformity/gradientindices. The total treatment time of the robotic radiosurgery treatments can be reduced by selecting nodes with more efficient robot traversal paths, while maintaining planquality.

Structural bases for blockade and activation of BK channels by Ba2+ ions.

We studied the impact of Ba2+ ions on the function and structure of large conductance potassium (BK) channels. Ion composition has played a crucial role in the physiological studies of BK channels due to their ability to couple ion composition and membrane voltage signaling. Unlike Ca2+, which activates BK channels through all Regulator of K + Conductance (RCK) domains, Ba2+ has been described as specifically interacting with the RCK2 domain. It has been shown that Ba2+ also blocks potassium permeation by binding to the channel's selectivity filter. The Cryo-EM structure of the Aplysia BK channel in the presence of high concentration Ba2+ here presented (PDBID: 7RJT) revealed that Ba2+ occupies the K+ S3 site in the selectivity filter. Densities attributed to K+ ions were observed at sites S2 and S4. Ba2+ ions were also found bound to the high-affinity Ca2+ binding sites RCK1 and RCK2, which agrees with functional work suggesting that the Ba2+ increases open probability through the Ca2+ bowl site (RCK2). A comparative analysis with a second structure here presented (PDBID: 7RK6), obtained without additional Ba2+, shows localized changes between the RCK1 and RCK2 domains, suggestive of coordinated dynamics between the RCK ion binding sites with possible relevance for the activation/blockade of the channel. The observed densities attributed to Ba2+ at RCK1 and RCK2 sites and the selectivity filter contribute to a deeper understanding of the structural basis for Ba2+'s dual role in BK channel modulation, adding to the existing knowledge in this field.