X-ray Crystallography Research Articles

Perspectives Toward an Integrative Structural Biology Pipeline With Atomic Force Microscopy TopographicImages.

After the recent double revolutions in structural biology, which include the use of direct detectors for cryo-electron microscopy resulting in a significant improvement in the expected resolution of large macromolecule structures, and the advent of AlphaFold which allows for near-accurate prediction of any protein structures, the field of structural biology is now pursuing more ambitious targets, including several MDa assemblies. But complex target systems cannot be tackled using a single biophysical technique. The field of integrative structural biology has emerged as a global solution. The aim is to integrate data from multiple complementary techniques to produce a final three-dimensional model that cannot be obtained from any single technique. The absence of atomic force microscopy data from integrative structural biology platforms is not necessarily due to its nm resolution, as opposed to Å resolution for x-ray crystallography, nuclear magnetic resonance, or electron microscopy. Rather a significant issue was that the AFM topographic data lacked interpretability. Fortunately, with the introduction of the AFM-Assembly pipeline and other similar tools, it is now possible to integrate AFM topographic data into integrative modeling platforms. The advantages of single molecule techniques, such as AFM, include the ability to confirm experimentally any assembled molecular models or to produce alternative conformations that mimic the inherent flexibility of large proteins or complexes. The review begins with a brief overview of the historical developments of AFM data in structural biology, followed by an examination of the strengths and limitations of AFM imaging, which have hindered its integration into modern modeling platforms. This review discusses the correction and improvement of AFM topographic images, as well as the principles behind the AFM-Assembly pipeline. It also presents and discusses a series of challenges that need to be addressed in order to improve the incorporation of AFM data into integrative modeling platform.

Understanding Cation–Anion Ionic Bonding in Tetramethylammonium Salts: Insights from Density Functional Theory and X-ray Crystallography

Understanding Cation–Anion Ionic Bonding in Tetramethylammonium Salts: Insights from Density Functional Theory and X-ray Crystallography

Synthesis, Structure, and Optical Property of Tris(biaryldiyl)metal Complexes Consisting of Group 9 Elements.

We report the synthesis and characterization of tris(biphenyl-2,2'-diyl)metal complexes of trivalent group 9 elements (1M, M = Co, Rh, Ir) and their nonplanarly π-extended analogs, tris(1,1'-binaphthyl-2,2'-diyl)metal complexes (2M, M = Rh, Ir). Single crystal X-ray crystallography reveals the distorted octahedral geometry with an approximate C3 symmetry of trianionic complexes 1M (M = Co, Rh, Ir) and 2M (M = Rh, Ir), which are contacted by three Li+ ions in the crystal. Complex 1Ir exhibits yellow luminescence in THF with a photoluminescence quantum yield (ΦPL) of up to 0.73, along with a distinctive photophysical property, namely, a concentration dependence of the emission wavelength from 530 to 580 nm. This is a characteristic property of 1Ir and has not been observed in its isoelectronic analog, tris(2-phenylpyridinato)iridium(III) (Ir(ppy)3). The concentration-dependent optical properties originate from the dissociation equilibria of Li+ ions from the anionic chromophore. Complex 2Ir also exhibits luminescence at 715 nm in THF, with a notable bathochromic shift from 1Ir through the π-extension. The findings offer insights into the photophysical properties of homoleptic organo-transition metal complexes, providing the foundation for the design of related transition metal complexes.

Beautoide A, an Anti-Osteoclastogenic Sterol from Beauveria sp. NBUF147 Associated with an Irciniidae Sponge from the Marine Mesophotic Zone.

Mesophotic coral ecosystems (MCEs), located at depths ranging from 30-150 m, host some of the most diverse yet least explored marine bioresources, particularly significant for the discovery of new bioactive molecules. The fungus Beauveria sp. NBUF147, associated with an Irciniidae sponge from the mesophotic zone at a depth of 82 m, underwent chemical investigation that led to the identification of one new sterol, beautoide A (1), and one reported sterol, 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (2). Their structures were determined from analysis of spectroscopic data and X-ray crystallography. Evaluation of biological activity in prednisolone-induced osteoporotic zebrafish showed that 1 was anti-osteoclastogenic in vivo at 3.0 μM.

Revisiting the Cyclocephagenols via Astragalus condensatus: Structural Insights and Configurational Revision.

The phytochemical investigation of the MeOH extract of Astragalus condensatus roots led to the discovery of a new tetrahydropyran cycloartane-type triterpenoid, astracondensatol A (1), alongside six known cyclocephagenol derivatives (2, 3, 20, 32, 35, and 36). Elucidation of their structures involved 1D and 2D-NMR spectroscopy and mass data analysis. Upon comparing NMR spectroscopic data with prior literature, several carbon shift anomalies, particularly at C-24, prompted a reevaluation using quantum chemical calculations, resulting in the revision of the 24S to 24R absolute configuration for compound 2 and 38 other reported cyclocephagenol-type triterpenoids. X-ray crystallography data further supported the analysis in establishing the absolute configuration of compound 2. Ambiguous NOE correlations and publication bias could have played a significant role in miss-assigning the C-24 absolute configuration in tetrahydropyran cycloartane-type triterpenoids.

Palladium-Catalyzed Acetoxylation of γ-Dehydro-aryl-himachalene: The Synthesis of a Novel Allylic Acetoxylated Sesquiterpene and a π-Allyl Palladium(II) Complex

Allylic oxygenated derivatives of himachalenes are highly valued molecules due to their potential applications in perfumery, cosmetics, and pharmaceuticals. Previous attempts at catalyzed allylic oxidation of himachalenes led to the formation of a very stable η3-allyl palladium complex, preventing any further reaction development. Herein, we present the first successful palladium-catalyzed synthesis of a novel allylic acetoxylated derivative of himachalenes. This reaction was achieved by incorporating an aromatic ring into the substrate structure. The resulting intermediate complex was isolated and characterized using nuclear magnetic resonance spectroscopy and X-ray crystallography. Density functional theory (DFT) calculations were performed to compare the reactivity of the newly synthesized complex with previously reported ones. The theoretical results confirm that the introduction of an aromatic ring enhances the reactivity of the η³-allyl palladium complex, thereby facilitating the desired transformation.

The Dialuminene AriPr8AlAlAriPr8 (AriPr8=C6H-2,6-(C6H2-2,4,6-iPr3)2-3,5-iPr2).

Careful analysis of the crystals formed in the reduction of AriPr8AlI2 (AriPr8=C6H-2,6-(C6H2-2,4,6-iPr3)2-3,5-iPr2) with sodium on sodium chloride showed them to contain the long sought-after dialuminene AriPr8AlAlAriPr8 (1) that forms alongside the previously characterized alanediyl :AlAriPr8. The single crystal X-ray structure of 1 revealed a nearly planar, trans-bent C(ipso)AlAlC(ipso) core with an Al-Al distance of 2.648(2) Å. The molecular and electronic structure of 1 are consistent with an Al-Al double dative interaction augmented with diradical character and stabilized by dispersion interactions. Density functional theory calculations showed that the reactivity of :AlAriPr8 with dihydrogen involves 1, not :AlAriPr8, as the reactive species. In contrast, the reaction of :AlAriPr8 with ethylene gave two products, the 1,4-dialuminacyclohexane AriPr8Al(C2H4)2AlAriPr8 (2) and the aluminacyclopentane AriPr8Al(C4H8) (3), that can both form from the aluminacyclopropane intermediate AriPr8Al(C2H4). Although the [2+2+2] cycloaddition of 1 with two equivalents of ethylene was also calculated to be exergonic, it is likely to be kinetically blocked by the numerous isopropyl substituents surrounding the Al-Al bond. Attempts to fine-tune the steric bulk of the terphenyl ligand to allow stronger Al-Al bonding were unsuccessful, leading to the isolation of the sodium salt of a cyclotrialuminene, Na2[AlAriPr6]3 (4), instead of AriPr6AlAlAriPr6.

A Double-walled Noncovalent Carbon Nanotube by Columnar Packing of Nanotube Fragments.

Herein, we report the synthesis of double-walled noncovalent carbon nanotubes (CNTs) through host-guest complexation of nanotube fragments and tube-forming crystal engineering. As the smallest fragment of double-walled CNTs, a host-guest complex of perfluorocycloparaphenylene (PFCPP) and carbon nanobelt (CNB) was synthesized by mixing them in solvents. The immediate complexation of the PF[12]CPP⊃(6,6)CNB complex with a remarkably high association constant (Ka) of 2×105 L/mol was observed. Time-dependent 1H NMR and thermogravimetry measurements revealed that the stability of (6,6)CNB was significantly improved by encapsulation. X-ray crystallography confirmed the robust belt-in-ring structure of this complex. As indicated by the short distance between PF[12]CPP and (6,6)CNB (2.8 Å), intermolecular orbital interactions exist between the belt and the ring, which were further supported by theoretical calculation and phosphorescence quenching experiments. While the PF[12]CPP⊃(6,6)CNB complex adopts various crystal packing structures, chloroform was discovered to be a magic "glue" solvent inducing one-dimensional alignment of the PF[12]CPP⊃(6,6)CNB complex to build an unprecedented double-walled noncovalent CNT structure.

Structural and theoretical insights into the influence of thiocyanate ligands on divalent metal complexes with terpyridine derivatives

Four complexes with terpyridine derivative, thiocyanate anion, and selected d-electron metals were studied. The obtained complexes of the formulation [M(ftpy)(SCN)2(CH3OH)]•DMF (M = Mn (1), Co (2), Ni (3)) and [Cu(ftpy)(NCS)2]•H2O (4), where ftpy = 4′‐(furan-2-yl)-2,2′:6′,2′′‐terpyridine, were characterized using FTIR and UV–Vis spectroscopies, magnetic studies, elemental analysis, and single crystal X-ray crystallography. All compounds crystallize in a triclinic crystal system with space group P1¯ and 1–3 are isomorphous and isostructural. The effect of the modification of the terpyridine ligand and the presence of the thiocyanate ligand on the structure has been verified. Interesting dependence was found in the structure of compound 4, where the two thiocyanate ligands are differently coordinated, one via N and the other via S, and the formation of complex dimers connected through hydrogen bonds is observed. The π-stacking interactions and the resulting solid-state architectures of compounds 1–4 were inspected through a combination of DFT calculations, QTAIM, and NCIPlot analyses, and the stability of the complexes was studied by UV–Vis spectroscopy in various solvents.

“Seeing Is Believing”: How Neutron Crystallography Informs Enzyme Mechanisms by Visualizing Unique Water Species

Hydrogen is the lightest atom and composes approximately half of the atomic content in macromolecules, yet their location can only be inferred or predicted in most macromolecular structures. This is because hydrogen can rarely be directly observed by the most common structure determination techniques (such as X-ray crystallography and electron cryomicroscopy). However, knowledge of hydrogen atom positions, especially for enzymes, can reveal protonation states of titratable active site residues, hydrogen bonding patterns, and the orientation of water molecules. Though we know they are present, this vital layer of information, which can inform a myriad of biological processes, is frustratingly invisible to us. The good news is that, even at modest resolution, neutron crystallography (NC) can reveal this layer and has emerged this century as a powerful tool to elucidate enzyme catalytic mechanisms. Due to its strong and coherent scattering of neutrons, incorporation of deuterium into the protein crystal amplifies the power of NC. This is especially true when solvation and the specific participation of key water molecules are crucial for catalysis. Neutron data allow the modeling of all three atoms in water molecules and have even revealed previously unobserved and unique species such as hydronium (D3O+) and deuteroxide (OD−) ions as well as lone deuterons (D+). Herein, we briefly review why neutrons are ideal probes for identifying catalytically important water molecules and these unique water-like species, limitations in interpretation, and four vignettes of enzyme success stories from disparate research groups. One of these groups was that of Dr. Chris G. Dealwis, who died unexpectedly in 2022. As a memorial appreciation of his scientific career, we will also highlight his interest and contributions to the neutron crystallography field. As both the authors were mentored by Chris, we feel we have a unique perspective on his love of molecular structure and admiration for neutrons as a tool to query those structures.

The H2Sxmacropa Series: Increasing the Chemical Softness of H2macropa with Sulfur Atoms to Chelate Radiometals [213Bi]Bi3+ and [203Pb]Pb2+ for Radiopharmaceutical Applications.

The effects of replacing nitrogen with sulfur atoms in the 18-membered macrocycle of the H2macropa chelator on the binding affinity and stability of "intermediate" (radio)metal [203Pb]Pb2+ and [213Bi]Bi3+ complexes are investigated. The 1,4,10,13-tetraoxo-7,16-diazacyclooctadecane backbone was replaced with derivatives containing sulfur in the 1,4- or the 1,4,10,13-positions to yield the novel chelators H2S2macropa (N4O4S2) and H2S4macropa (N4O2S4), respectively. Trends on the nat/203Pb- and nat/213Bi-complex stability constants, coordination chemistry, radiolabeling, and kinetic inertness were assessed via potentiometric titrations, UV-vis spectroscopy, NMR spectroscopy, X-ray crystallography and density functional theory (DFT) calculations. 1H-207Pb NMR spectroscopy confirmed the involvement of backbone S and/or O donors in the metal coordination sphere. Overall, the trend demonstrated that increasing the softness of the donor atoms within the ligand backbone decreased the thermodynamic stability and kinetic inertness of both the Pb2+ and Bi3+ complexes. Conversely, DFT calculations with mock compounds dimethyl ether (DME) and dimethyl sulfide (DMS) demonstrated enhanced affinity of the S atom to both Pb2+ and Bi3+ with DMS compared to DME evinced by large ΔG° values for both Pb2+ and Bi3+ complexes. The decreased stability of Pb/Bi-Sxmacropa (x = 0, 2, 4) upon increased sulfur atom incorporation may be a result of the increased steric strain within the macrocyclic backbone upon sulfur atom introduction. Nonetheless, [203Pb]Pb2+ and [213Bi]Bi3+ labeling (pH = 7, 30 min reaction time; 10-4-10-8 M chelator) resulted in both S2macropa2- and macropa2- attaining similarly high radiolabeling efficiency. Meanwhile, S4macropa2- only possessed the ability to complex [213Bi]Bi3+. Both [203Pb][Pb(macropa)] and [203Pb][Pb(S2macropa)] remained greater than 97% intact when challenged against human serum over 72 h. The results of this study reveal the effects of incorporating sulfur donor atoms into macrocyclic chelators for [203Pb]Pb2+ and [213Bi]Bi3+ radiopharmaceuticals.

Albumins constrainting the conformation of mitochondria-targeted photosensitizers for tumor-specific photodynamic therapy

Tumor ablation Preclinical organelle-targeted phototherapies have effectively achieved tumor photoablation for regenerative biomedical applications in cancer therapies. However, engineering effective phototherapy drugs with precise tumor-localization targeting and organelle direction remains challenging. Herein, we report a albumins constrainting mitochondrial-targeted photosensitizer nanoparticles (PSs@BSAs) for tumor-specific photodynamic therapy. X-ray crystallography elucidates the two-stage assembly mechanism of PSs@BSAs. Femtosecond transient absorption spectroscopy and quantum mechanical calculations reveal the implications of conformational dynamics at the excited state. PSs@BSAs can efficiently disable mitochondrial activity, and further disrupt tumor angiogenesis based on the photodynamic effect. This triggers a metabolic and oxidative stress crisis to facilitate photoablation of solid tumor and antitumor metastasis. The study fully elucidates the interdisciplinary issues of chemistry, physics, and biological interfaces, thereby opening new horizons to inspire the engineering of organelle-targeted tumor-specific photosensitizers for biomedical applications.

PnictogenIIIDications Supported by BZIMPY Ligands.

Two homologous series of pnictogen(III) dications, stabilized by 2,6-bis(benzimidazole-2-yl)pyridine ligands have been prepared. Both series contain PnIII-X moieties (Pn = P, As, Sb, Bi; X = Cl or Ph) and have been fully characterized using spectroscopic methods including X-ray crystallography. The Lewis acidity of these compounds has also been probed by computational methods; the results suggest that the dictations are strong Lewis acids, with the PnCl2+ compounds being more acidic than the PnPh2+ compounds, and with Lewis acidity increasing from P to Bi, in both series. The PhP2+-containing compound was also found to be a versatile PIII transfer reagent, leading to new synthetic routes for various PhP-containing compounds. The redox chemistry of all compounds has also been probed using cyclic voltammetry and chemical reductions. In some cases the resulting PnI moieties could be trapped using diazabutadienes.

Exploring Charge Redistribution at the Cu/Co6Se8 Interface.

This study investigates the electronic interactions and charge redistribution at the dopant-support interface using a Cu/Co6Se8 cluster construct. Specifically, the redox cluster series [Cu3Co6Se8L6]n ([1-Cu3]n; n = 0, -1, -2, -3; L = Ph2PNTol-, Ph = phenyl, Tol = p-tolyl) spanning four distinct oxidation states is synthesized and characterized using a multitude of techniques, including multinuclear NMR, UV-vis, XANES, and X-ray crystallography. Structural investigations indicate that the clusters are isostructural and chiral, adopting a pseudo-D3 symmetry. Paramagnetic 31P NMR spectroscopy and solution-phase magnetic measurements together with DFT calculations are employed to interrogate the electronic structure and spin-state changes across the [1-Cu3]3- to 1-Cu3 redox series, revealing that the copper edge sites retain a +1 oxidation state while the Co/Se core becomes increasingly oxidized, yielding a highly zwitterionic cluster.

Evidence for the Quercetin Binding Site of Glycogen Phosphorylase as a Target for Liver-Isoform-Selective Inhibitors against Glioblastoma: Investigation of Flavanols Epigallocatechin Gallate and Epigallocatechin.

Glycogen phosphorylase (GP) is the rate-determining enzyme in glycogenolysis, and its druggability has been extensively studied over the years for the development of therapeutics against type 2 diabetes (T2D) and, more recently, cancer. However, the conservation of binding sites between the liver and muscle isoforms makes the inhibitor selectivity challenging. Using a combination of kinetic, crystallographic, modeling, and cellular studies, we have probed the binding of dietary flavonoids epigallocatechin gallate (EGCG) and epigallocatechin (EGC) to GP isoforms. The structures of rmGPb-EGCG and rmGPb-EGC complexes were determined by X-ray crystallography, showing binding at the quercetin binding site (QBS) in agreement with kinetic studies that revealed both compounds as noncompetitive inhibitors of GP, with EGCG also causing a significant reduction in cell viability and migration of U87-MG glioblastoma cells. Interestingly, EGCG exhibits different binding modes to GP isoforms, revealing QBS as a promising site for GP targeting, offering new opportunities for the design of liver-selective GP inhibitors.

Synthesis Techniques and Biomedical Applications of Cyclodextrin Metal-Organic Frameworks.

Cyclodextrin Metal-Organic Frameworks (CD MOFs) represent an innovative class of materials with remarkable properties and a broad range of applications. This review provides a comprehensive overview of the synthesis techniques, structural characterization, and diverse applications of CD-MOFs. By combining cyclodextrins (CDs) with metal-organic frameworks (MOFs), CD-MOFs are developed with enhanced functionality. The synthesis methods, including various metal sources, coordination modes, and post-synthesis modifications, are discussed alongside advanced structural characterization techniques like X-ray crystallography and spectroscopic methods. The unique characteristics of CD-MOFs, such as high specific surface area, tunable porosity, and customizable chemical structure, make them exceptional candidates for applications in gas adsorption, drug delivery, catalysis, sensing, and environmental remediation. Notably, CD-MOFs show significant promise as nanocarriers in drug delivery systems, offering improved therapeutic outcomes due to their efficient encapsulation and controlled release capabilities. The review highlights recent advancements and underscores the potential impact of CD-MOFs in driving future innovations across various scientific fields.

Skeletal Editing of Mechanically Interlocked Molecules: Nitrogen Atom Deletion from Crown Ether-Dibenzylammonium Rotaxanes.

Removing the nitrogen atom from secondary amines while simultaneously linking the remaining fragments is a powerful form of late-stage skeletal editing. Here, we report its use for the deletion of the nitrogen atom of the dibenzylammonium template used to assemble crown ether rotaxanes. The reaction uses an anomeric amide that activates secondary amines to generate a carbon-carbon bond that replaces the amine nitrogen. Despite the potential for dethreading of the intermediate diradical pair, the nitrogen atom was successfully deleted from a series of rotaxane axles as long as the macrocycle could access coconformations that did not inhibit the reaction of the amine group. The skeletally edited interlocked molecules were obtained directly from the parent crown ether-dibenzylammonium rotaxanes in modest yields (23-36%) and characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography. One skeletally edited rotaxane shows a network of weak CH···O hydrogen bonds between the crown ether and benzylic methylene groups of the axle in the solid state, in place of the crown ether-ammonium binding motif used to assemble the parent, unedited, rotaxane.

Evidence of short chains in liquid sulfur.

High energy x-ray pair distribution function measurements show the average coordination number of the first shell in liquid sulfur is 1.86 ± 0.04 across the λ-transition, not precisely 2.0 as widely accepted. This indicates that upon melting, liquid sulfur does not comprise solely of S8 rings but also possesses a significant number of short chains. Intensities of the pre-peak and first diffraction peak of the x-ray structure factor and third peak height of the pair distribution function all show deviations at the λ-transition temperature Tλ, associated with the break-up of S8 rings and the start of oligomer polymerization. A significant number of non-bonded or loosely bonded "interstitial atoms," with an average coordination number of 0.20 ± 0.005, are also observed in the so-called "forbidden zone" between the first and second shells upon melting. The number of interstitial atoms is found to decrease to a minimum at the λ-transition, but the majority persist into the high temperature polymerized liquid. The existence of short chains and nearby interstitial atoms represent the two main factors required to initiate the S8-ring to chain transition, as proposed by recent molecular dynamics simulations.

Pyrrolizwilline, a unique bacterial alkaloid assembled by a nonribosomal peptide synthetase and non-enzymatic dimerization.

Pyrrolizidine alkaloids (PAs) are a structurally diverse group of heterocyclic specialized metabolites characterized by a core structure comprising a hexahydro-1H-pyrrolizine. PAs are synthesized through two main pathways. In plants, assembly occurs via a homospermidine synthase, and in bacteria, through combined action of a nonribosomal peptide synthetase and a Baeyer-Villiger monooxygenase. While the toxic properties of plant-derived PAs and their prevalence in animal and human foods have been extensively studied, the biological roles and biosynthesis of more complex bacterial PAs are not well understood. Here, we report the identification and characterization of a bacterial biosynthetic gene cluster fromXenorhabdus hominickii,xhpA-G, which is responsible for producing the PA pseudo-dimer pyrrolizwilline. Analysis ofX. hominickiipromoter exchange mutants together with heterologous expression ofxhpA-GinE. coli, revealed a set of pathway intermediates, two of which were chemically synthesized, as well as multiple derivatives. This information was leveraged to propose a detailed biosynthetic pathway to pyrrolizwilline. Furthermore, we have characterized the hydrolase XhpG, the key enzyme in the conversion of the pathway intermediate pyrrolizixenamide to pyrrolizwilline, using X-ray crystallography and small-angle X-ray scattering (SAXS).

Revealing protein structures: crystallization of protein-ligand complexes - co-crystallization and crystalsoaking.

Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.