Structure Of Complex Research Articles

Phosphonate-based iron complex for a cost-effective and long cycling aqueous iron redox flow battery

A promising metal-organic complex, iron (Fe)-NTMPA2, consisting of Fe(III) chloride and nitrilotri-(methylphosphonic acid) (NTMPA), is designed for use in aqueous iron redox flow batteries. A full-cell testing, where a concentrated Fe-NTMPA2 anolyte (0.67 M) is paired with a Fe-CN catholyte, demonstrates exceptional cycling stability over 1000 charge/discharge cycles, and noteworthy performances, including 96% capacity utilization, a minimal capacity fade rate of 0.0013% per cycle (1.3% over 1,000 cycles), high Coulombic efficiency and energy efficiency near 100% and 87%, respectively, all achieved under a current density of 20 mA·cm-². Furthermore, density functional theory unveils two potential coordination structures for Fe-NTMPA2 complexes, improving the understanding between the ligand coordination environment and electron transfer kinetics. When paired with a high redox potential Fe-Dcbpy/CN catholyte, 2,2′-bipyridine-4,4′-dicarboxylic (Dcbpy) acid and cyanide (CN) ligands, Fe-NTMPA2 demonstrates a notably elevated cell voltage of 1 V, enabling a practical energy density of up to 9 Wh/L.

Geological structure and metallogeny of Precambrian sedimentary structures of western Aldan-Stanovy Shield

The article presents the results of the study by various geologists of the geological structure and prospects of ore-bearing trog complexes performing structures such as throgs, which were identified at different stages of geological research in the western part of the Aldan-Stanovy shield. The main types of traction complexes and their sections were determined. The nature of the occurrence of sedimentary (terrigenous), volcanogenicsedimentary and metavolcanic rocks performing throgs and forming throg complexes was established. The nature of the ratio of these rocks in the context of the trog complex in the studied trogs was described. The results of the study of trog complexes showed that they are generally characterized by facies variability, heterogeneity of metamorphism and dynamothermal processing of rocks (especially in the sides of trogs) with the formation of mylonites and cataclasites. It was established that the throgs are confined to three regional systems of discontinuous disturbances. Both consonantal and secant discontinuities were marked inside the throgs, defining their block tectonics. The results of determining the geological age of the rocks of the trogovoi complex were presented. The trog structures are characterized by ore mineralization associated with the processes of sedimentation (ferruginous quartzites, copper sandstones) and volcanogenic ore formation (copper-crusted and polymetallic mineralization), as well as with the processes of metamorphogenic-hydrothermal redistribution of ore matter (stratiform scheelite mineralization, ore-bearing quartz veins). It was also found that the metallogeny of the trog structures has not been studied sufficiently. Nevertheless, the majority of geologists who studied the trogovye complexes noted in their reports that Precambrian trogovye complexes can be considered as important and metallogenically promising geological objects in the western part of the Aldan-Stanovy Shield and deserve more in-depth study.

Substrate Selection Criteria in Regulated Intramembrane Proteolysis.

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aβ peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

Structure and digestibility of bovine casein complexes formed by enriching κ- and β-caseins in micellar casein concentrate together with minerals adjustment

Colloidal structure and infant in vitro gastrointestinal digestibility of casein complexes simulating caseins and minerals compositions of human casein micelles were studied. κ- and β-Caseins were fractionated from bovine micellar casein concentrate (MCC), and mixed with MCC to increase their ratios to 20% and 68%, followed by adding citrate (Cit), calcium (Ca), and inorganic phosphate (Pi) to obtain the casein complexes. The complexes enriched with κ- and β-caseins showed higher percentages of serum Ca, Pi and caseins, larger particle size, and looser internal structure. During gastric digestion, the complexes enriched with κ- and β-caseins formed smaller and looser coagula, which promoted degradation of intact caseins and formation of free amino groups and small peptides. During initial intestinal digestion, the complexes enriched with κ- and β-caseins formed more free amino groups and smaller peptides. These results offered a potential strategy to form human casein micelles analogues for use in infant formula.

Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq

Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.

Modification mechanism of soybean protein isolate-soluble soy polysaccharide complex by EGCG through covalent and non-covalent interaction: Structural, interfacial, and functional properties

Soybean protein isolate was modified with polysaccharides and polyphenols to prepare a natural emulsifier with antioxidant capacity. Physicochemical, structural, interfacial, and functional properties of SPI-SSPS complex were investigated after covalent and non-covalent interacted with EGCG. SPI-SSPS-EGCG ternary complex with low EGCG concentrations (0.0625 and 0.125 mg/mL) showed a significant increase in absolute potential value and a decrease in turbidity. EGCG destroyed the original rigid structure of SPI-SSPS complex, and the covalent complexes had an ordered structure, while the non-covalent interaction resulted in disordered. The ternary complex with high EGCG concentrations (0.25 and 0.5 mg/mL) exhibited stronger EGCG binding capacity and lower surface hydrophobicity, which in turn affected its interfacial properties. The EAI and ESI of SPI-SSPS-EGCG covalent complex increased significantly, while the non-covalent complex had a significant change in EAI but no significant change in ESI with increasing EGCG concentration. The ternary complex showed significantly enhanced antioxidant capacity. The SPI-SSPS-EGCG ternary complex, with excellent antioxidant capacity and emulsifying property, making it suitable for emulsion delivery systems.

Structure of the human Bre1 complex bound to the nucleosome

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0–6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

Abstract 2348: A global multiscale map of protein assemblies from integration of protein interactions and images

Abstract Cells regulate growth and function through a hierarchical structure of subcellular protein assemblies, in which alterations can result in cellular dysfunction leading to disease such as cancer. Much of this structure remains uncharted, resulting in recent efforts to map subcellular organization at different physical scales. Here, we report a global architectural map of human cancer cell protein assemblies spanning 10−9 to 10−5 nm, based on integration of near-proteome-wide affinity purification mass spectrometry-based protein interactions and immunofluorescent imaging in U-2 OS osteosarcoma cancer cells. The U-2 OS multi-scale integrated cell map places >5000 proteins into 270 distinct subcellular protein assemblies across biological scales, representing approximately half of expressed proteins. There are known organelles and protein complexes recovered in the map, as well as 152 putative assemblies, such as a putative interferon signaling complex consisting of a serine protease and STAT transcription factors. The map also incorporates 128 previously uncharacterized proteins into protein assemblies, such as C18orf21 association with a canonical RNAse complex. Protein subsystems in the U-2 OS map were evaluated for the potential for downstream integrative structural modeling, where available structural data (e.g. crosslinking, Protein Data Bank structures, AlphaFold predictions) are combined in a Bayesian framework to model the physical structures of protein complexes. Using this framework, we created an integrated structural model of new proteins interacting with the Rag-Ragulator complex, which regulates MAPK and mTOR signaling. In summary, the global proteome architecture map provides a resource for cellular biology discovery and the systematic determination of protein functions and structures. Citation Format: Leah V. Schaffer, Mengzhou Hu, Edward L. Huttlin, Gege Qian, Abantika Pal, Neelesh Soni, Andrew Latham, Laura Pontano Vaites, Trang Le, Yue Qin, Christopher Churas, Dexter Pratt, Ignacia Echeverria, Andrej Sali, J Wade Harper, Steven P. Gygi, Emma Lundberg, Trey Ideker. A global multiscale map of protein assemblies from integration of protein interactions and images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2348.

Synthesis, characterization, and structures of mononuclear and phenoxo-/acetato-bridged trinuclear zinc(II) complexes from salan ligands: Study of their hydrolytic activity

Synthesis of one mononuclear [ZnL1] (1) and two trinuclear phenoxo-/acetato-bridged zinc(II) complexes [Zn3Cl2(L2)2] (2) and [Zn3(OAc)2(L1)2] (3) containing tetradentate diaminobis(phenolato) “salan” ligands are reported. The ligand precursors and their corresponding zinc complexes are characterized by elemental analysis, NMR, IR, and UV–vis spectroscopy. Molecular structures of complexes 2 and 3 are solved by single-crystal X-ray crystallography, while the fate of the complexes in solution is assessed by ESI-MS spectrometry analysis. All complexes (1–3) were studied for their phosphoesterase activity by using para-nitrophenyl phosphate (PNPP) and bis(2,4–dinitrophenyl) phosphate (BNPP) as substrates. While complex 1 has no activity for these substrates, complex 3 effectively hydrolyses PNPP (1.6 × 10−3 s−1), and complex 2, which is less reactive than 3, displays comparable activities towards both substrates (2.0 × 10−4 s−1 and 5.4 × 10−4 s−1, respectively). The importance of evaluating the speciation of Zn-species present for the understanding of the mechanism of the phosphoesterase reaction is discussed.

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

Cryo-EM structures of RAD51 assembled on nucleosomes containing a DSB site.

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.

Can Iron-Porphyrins Behave as Single-Molecule Magnets?

The study of magnetic properties, especially the magnetic anisotropy of iron-porphyrin complexes employing multiconfigurational methods, is quite challenging due to many strongly correlated electrons in nearly degenerate orbitals. However, a prerequisite for observing the magnetic anisotropy and slow magnetization relaxation, the zero-field splitting parameter, D, was experimentally observed decades ago for halide-based axially ligated penta-coordinate Fe(III)-porphyrins. In these complexes, the signs of D were reported mostly as positive; in a few cases, inconclusive signs of the D parameter were also mentioned. However, no ab initio calculations have been reported to shed light on this. Deciphering the electronic structure of these penta-coordinated complexes employing the complete active space self-consistent field method and N-electron valence second-order perturbation theory, we confirm the positive D values. However, a negative D value is highly desired to observe the single-molecule magnet properties without an external magnetic field, which we observed in the Fe(II)-porphyrin complexes with axial imidazole ligands instead of halide ligands. The detailed analysis of the multireference wave functions unravels the role of axial ligands in determining the sign and magnitude of the D parameters.

ФОРМУВАННЯ МОДЕЛЕЙ КЛАСИФІКАЦІЇ НЕВИЗНАЧЕНИХ ДАНИХ ПРОЦЕДУРАМИ РЕДУКЦІЇ І КАППА СТАТИСТИКИ

The article is devoted to the development of mathematical models for the classi-fication of uncertain data represented by fuzzy values and certainty factors CF(A). Diagnostic pattern formation procedures use modified Hamming networks (MHN), as well as reduction methods and Cohen's kappa statistics. At the same time, the limit-ing dimensions and composition of the parameters of the classification model are de-termined, which ensure the established probabilistic requirements for the reliability of the calculation results. The model space reduction procedure and the structure of the software complex for diagnosing uncertain data are presented. An example of a clas-sification model based on fuzzy data is the task of identifying the authors of Ukrain-ian-language texts. The classification task for data in CF(A) format corresponds to candidate selection. The results of the numerical modeling made it possible to estab-lish the effectiveness, reliability and efficiency of the proposed procedures for the formation of reliable classification models with uncertain data.

Ion pair induced supramolecular assembly for development of novel pyridine 2, 6 diamide spacer acyl hydrazone-based derivatives for selective sensing of ZnCl2 and AlCl3

The work reports the development of a novel pyridine 2, 6 diamide spacer acyl hydrazone-based dual sensor H2L via two intermediates 1 and 2. The sensor as well as intermediates have been spectroscopically characterized. The structure of intermediate 1 is validated by its single crystal data analysis. As H2L bears both amide and acyl hydrazone moiety which have selective detection ability of anion and cation respectively, after rigorous characterization, the sensors are exposed to check their dual binding behavior. The results of several spectroscopic studies disclose the fact that H2L can optically and fluorometrically detect Al (III) and Zn (II) among several competitive cations and Cl− among competitive anions in semi-aqueous and aqueous medium respectively. Interestingly in a 9:1 DMSO-water medium, the probe can act as an ion pair sensor and in this medium, the probe can simultaneously detect Zn (II)/Cl- and Al (III)/Cl- forming H2L-ZnCl2, H2L-AlCl3 adduct. The 1:1 probe-analyte ion-pair binding is confirmed by NMR and mass spectral study. The Density Functional Theory (DFT) study optimizes the structures of the probe-analyte complexes. Impressively the probe has the potential to detect and quantify the Al (III) from market-available drug samples.

Implementation of pedagogical integration in the structure of the university complex

The purpose of the work is to describe the conduct and results of an experiment to confi rm pedagogical integration in a university complex.The subject of the study is the joint activity of teaching staff of educational organizations of the university complex. The relevance is due to the integrative orientation of modern education.Scientific novelty consists in obtaining new knowledge formed as a result of the application of methods developed in the study of the organization of educational activities in the university complex, providing pedagogical integration.The practical significance (public and scientific) of the research is aimed at ensuring the joint integrative activity of pedagogical and scientific-pedagogical workers of organizations of secondary vocational and higher education in the structure of the university complex.Scientific and pedagogical works, socio-pedagogical and qualimetric methods were used in the work.The conducted experiment showed the achieved positive long-term result of the study, which allows to systematize the work of pedagogical collectives of organizations, individualize the educational trajectories of students, eliminate duplication of the content of professional educational programs, reduce the duration of training in educational programs of secondary vocational and higher education.Pedagogical integration contributes to the successful implementation of educational programs through the effective use of pedagogical technologies.

SpatialPPI: Three-dimensional space protein-protein interaction prediction with AlphaFold Multimer

Rapid advancements in protein sequencing technology have resulted in gaps between proteins with identified sequences and those with mapped structures. Although sequence-based predictions offer insights, they can be incomplete due to the absence of structural details. Conversely, structure-based methods face challenges with respect to newly sequenced proteins. The AlphaFold Multimer has remarkable accuracy in predicting the structure of protein complexes. However, it cannot distinguish whether the input protein sequences can interact. Nonetheless, by analyzing the information in the models predicted by the AlphaFold Multimer, we propose a highly accurate method for predicting protein interactions. This study focuses on the use of deep neural networks, specifically to analyze protein complex structures predicted by the AlphaFold Multimer. By transforming atomic coordinates and utilizing sophisticated image-processing techniques, vital 3D structural details were extracted from protein complexes. Recognizing the significance of evaluating residue distances in protein interactions, this study leveraged image recognition approaches by integrating Densely Connected Convolutional Networks (DenseNet) and Deep Residual Network (ResNet) within 3D convolutional networks for protein 3D structure analysis. When benchmarked against leading protein-protein interaction prediction methods, such as SpeedPPI, D-script, DeepTrio, and PEPPI, our proposed method, named SpatialPPI, exhibited notable efficacy, emphasizing the promising role of 3D spatial processing in advancing the realm of structural biology. The SpatialPPI code is available at: https://github.com/ohuelab/SpatialPPI.

Structural insights into human organic cation transporter 1 transport and inhibition

The human organic cation transporter 1 (hOCT1), also known as SLC22A1, is integral to hepatic uptake of structurally diversified endogenous and exogenous organic cations, influencing both metabolism and drug pharmacokinetics. hOCT1 has been implicated in the therapeutic dynamics of many drugs, making interactions with hOCT1 a key consideration in novel drug development and drug–drug interactions. Notably, metformin, the frontline medication for type 2 diabetes, is a prominent hOCT1 substrate. Conversely, hOCT1 can be inhibited by agents such as spironolactone, a steroid analog inhibitor of the aldosterone receptor, necessitating a deep understanding of hOCT1–drug interactions in the development of new pharmacological treatments. Despite extensive study, specifics of hOCT1 transport and inhibition mechanisms remain elusive at the molecular level. Here, we present cryo-electron microscopy structures of the hOCT1-metformin complex in three distinct conformational states — outward open, outward occluded, and inward occluded as well as substrate-free hOCT1 in both partially and fully open states. We also present hOCT1 in complex with spironolactone in both outward and inward facing conformations. These structures provide atomic-level insights into the dynamic metformin transfer process via hOCT1 and the mechanism by which spironolactone inhibits it. Additionally, we identify a ‘YER’ motif critical for the conformational flexibility of hOCT1 and likely other SLC22 family transporters. Our findings significantly advance the understanding of hOCT1 molecular function and offer a foundational framework for the design of new therapeutic agents targeting this transporter.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

Functionalized graphene-oxide grids enable high-resolution cryo-EM structures of the SNF2h-nucleosome complex without crosslinking

Single-particle cryo-EM is widely used to determine enzyme-nucleosome complex structures. However, cryo-EM sample preparation remains challenging and inconsistent due to complex denaturation at the air-water interface (AWI). Here, to address this issue, we develop graphene-oxide-coated EM grids functionalized with either single-stranded DNA (ssDNA) or thiol-poly(acrylic acid-co-styrene) (TAASTY) co-polymer. These grids protect complexes between the chromatin remodeler SNF2h and nucleosomes from the AWI and facilitate collection of high-quality micrographs of intact SNF2h-nucleosome complexes in the absence of crosslinking. The data yields maps ranging from 2.3 to 3 Å in resolution. 3D variability analysis reveals nucleotide-state linked conformational changes in SNF2h bound to a nucleosome. In addition, the analysis provides structural evidence for asymmetric coordination between two SNF2h protomers acting on the same nucleosome. We envision these grids will enable similar detailed structural analyses for other enzyme-nucleosome complexes and possibly other protein-nucleic acid complexes in general.