Crystal Structure Determination Research Articles

Crystal Structure Determination from Powder Diffraction Patterns with Generative Machine Learning

Crystal Structure Determination from Powder Diffraction Patterns with Generative Machine Learning

Disulfide Bond Engineering of Soluble ACE2 for Thermal Stability Enhancement.

Although the primary pandemic of SARS-CoV-2 is over, there are concerns about the resurgence of the next wave of related viruses, including a wide range of variant viruses. The soluble ACE2 (sACE2) inhibits the SARS-CoV-2 spike protein ACE2 interaction and has potential as a variant-independent therapeutic against SARS-CoV-2. Here, we introduce novel disulfide bonds in the wild-type sACE2-Fc by structure-guided mutagenesis, aiming to improve its stability. The stability of each mutant was assessed by a thermal shift assay to screen mutants with increased thermal stability. As a result, we identified a mutant sACE2-Fc with a significantly increased melting temperature. X-ray crystal structure determination of the sACE2 mutant confirmed the correct formation of the designed disulfide bond, and there were no significant structural disturbances. We also proved that the thermostable sACE2-Fc preserved the spike protein binding affinity comparable to the wild-type sACE2-Fc in both molecular and cellular environments, suggesting its therapeutic potential.

Structural analysis of a ligand-triggered intermolecular disulfide switch in a major latex protein from opium poppy.

Several proteins from plant pathogenesis-related family 10 (PR10) are highly abundant in the latex of opium poppy and have recently been shown to play diverse and important roles in the biosynthesis of benzylisoquinoline alkaloids (BIAs). The recent determination of the first crystal structures of PR10-10 showed how large conformational changes in a surface loop and adjacent β-strand are coupled to the binding of BIA compounds to the central hydrophobic binding pocket. A more detailed analysis of these conformational changes is now reported to further clarify how ligand binding is coupled to the formation and cleavage of an intermolecular disulfide bond that is only sterically allowed when the BIA binding pocket is empty. To decouple ligand binding from disulfide-bond formation, each of the two highly conserved cysteine residues (Cys59 and Cys155) in PR10-10 was replaced with serine using site-directed mutagenesis. Crystal structures of the Cys59Ser mutant were determined in the presence of papaverine and in the absence of exogenous BIA compounds. A crystal structure of the Cys155Ser mutant was also determined in the absence ofexogenous BIA compounds. All three of these crystal structures reveal conformations similar to that of wild-type PR10-10 with bound BIA compounds. In the absence of exogenous BIA compounds, the Cys59Ser and Cys155Ser mutants appear to bind an unidentified ligand or mixture of ligands that was presumably introduced during expression of the proteins in Escherichia coli. The analysis of conformational changes triggered by the binding of BIA compounds suggests a molecular mechanism coupling ligand binding to the disruption of an intermolecular disulfide bond. This mechanism may be involved in the regulation of biosynthetic reactions in plants and possibly other organisms.

Kinetic and Catalytic Mechanism of the Methionine-Derived Glucosinolate Biosynthesis Enzyme Methylthioalkylmalate Synthase

In Brassica plants, methionine-derived aliphatic glucosinolates are chemically diverse natural products that serve as plant defense compounds, as well as molecules with dietary health-promoting effects. During their biosynthesis, methylthioalkylmalate synthase (MAMS) catalyzes the elongation reaction of the aliphatic chain. The MAMS catalyzed condensation of 4-methylthio-2-oxobutanoic acid (4-MTOB) and acetyl-CoA generates a 2-malate derivative that either enters the pathway for synthesis of C3-glucosinolates or undergoes additional extension reactions, which lead to C4- to C9-glucosinolates. Recent determination of the x-ray crystal structure of MAMS from Brassica juncea (Indian mustard) provided insight on the molecular evolution of MAMS, especially substrate specificity changes, from the leucine biosynthesis enzyme α-isopropylmalate synthase (IPMS) but left details of the reaction mechanism unanswered. Here we use the B. juncea MAMS2A (BjMAMS2A) isoform to analyze the kinetic and catalytic mechanism of this enzyme. Initial velocity studies indicate that MAMS follows an ordered bi bi kinetic mechanism, which based on the x-ray crystal structure, involves binding of 4-MTOB followed by acetyl-CoA. Examination of the pH-dependence of kcat and kcat/Km are consistent with acid/base catalysis. Site-directed mutagenesis of three residues originally proposed to function in the reaction mechanism - Arg89 (R89A, R89K, R89Q), Glu227 (E227A, E227D, E227Q), and His388 (H388A, H388N, H388Q, H388D, and H388E) - showed that only two mutants (E227Q and H388N) retained activity. Based on available structural and biochemical data, a revised reaction mechanism for MAMS-catalyzed elongation of methionine-derived aliphatic glucosinolates is proposed, which is likely also conserved in IPMS from leucine biosynthesis in plants and microbes.

Improvement of Single-Crystal Structures of Very Heavy Element Compounds by Refining Anomalous Dispersion Parameters.

Refining the anomalous dispersion parameters of the four uranium compounds NaUF5, NaU2F9, Cs2(UO2)TiO4, and Cs2(UO2)Ti2O6 gave insights into the crystallographic model improvement of very heavy atoms. We found that the values for the dispersive and absorptive parts, f' and f″, closely followed the X-ray absorption spectra on their L3, L2, and L1 edges. The obtained values are sensitive to the chemical environment at each crystallographically independent position. An incorrect treatment of the anomalous dispersion correction can lead to a wrong crystallographic model. The above-mentioned, already published structures were improved by this process. General guidelines were given for the crystal structure determination of very heavy compounds. When using Mo Kα radiation with uranium compounds, the proximity of its energy to the uranium L-edges causes a noticeable effect.

Deciphering the cytotoxic and antioxidant potential of 3,5-disubstituted isoxazole-linked benzimidazolone derivatives

A series of new isoxazole derivatives containing a benzimidazolone has been synthesized by cycloaddition reaction using various nitrile oxides. In all cases, these cycloadditions are completely chemo and regioselective. The structures of the new cycloadducts were characterized by 1H, 13C NMR spectroscopy, HRMS and confirmed in one case by a single crystal structure determination. The isoxazole compounds were evaluated for in vitro antioxidant activity using the DPPH and ABTS methods. As a result, compound 5a showed good antioxidant activity. The results of the acute toxicity study of the heterocycles studied by the oral route showed no deaths and no clinical signs of toxicity. Additionally, the Hirshfeld surface analysis and the molecular electrostatic potential using density functional theory (DFT) modeling at the B3LYP/6–31G(d,p) level. Hirshfeld surface analysis (dnorm surface and 2D-fingerprint plots) revealed the nature of intermolecular contacts. H⋯H interactions make the most significant contribution to crystal packing. The optimized structural parameters obtained through DFT calculations closely matched those determined via X-ray analysis, demonstrating strong agreement between theoretical predictions and experimental observations.

The influence of the pyridyl substituent in N-methyl-P,P-diphenyl-N-2-pyridinyl-phosphinous amide ligand on the coordination chemistry of group 6B metal carbonyl derivatives

The reaction of N-methyl-P,P-diphenyl-N-2-pyridinyl-phosphinous amide (1) with group 6B metal hexa, tetra, and tricarbonyls yielded the same octahedral complexes cis-M(CO)4[1-κ2P,Npy] (M = Cr(2), Mo(3), W(4)) in which the ligand chelates only in a bidentate fashion through its phosphorus atom and pyridyl N-atom. Compounds 1–4 were identified and characterized by multinuclear NMR spectroscopy (1H, 13C, 31P NMR), elemental analysis, IR and UV–Vis spectroscopy. Complexes 2–4 exhibited weak metal-to-ligand charge transfer transitions unaffected by solvent polarity. Crystal structure determination is carried out on complex 4.

Synthesis, characterization, and biological evaluation of novel cobalt(II) complexes with β-diketonates: crystal structure determination, BSA binding properties and molecular docking study.

In order to discover a new antibiotic drug with better or similar activity of the already existing drugs, a series of novel cobalt(II) complexes with β-diketonate as ligands is synthesized and tested on four strains of bacteria and four species of fungi. All compounds showed notable antimicrobial activityagainst all tested strains. More importantly, some cobalt(II) complexes displayed greater activity than ketoconazole. It is important to notice that on the tested strains Mucor mucedo and Penicillium italicum complex 2B showed five times better activity compared to ketoconazole, while complex 2D had two times better activity on Penicillium italicum strain compared to ketoconazole. Moreover, investigations with bovine serum albumin were performed. Investigations showed that the tested complexes have an appropriate affinity for binding to bovine serum albumin. In addition, the molecular docking study was performed to investigate more specifically the sites and binding mode of the tested cobalt(II) complexes with β-diketonate as ligands to bovine serum albumin, tyrosyl-tRNA synthetase, topoisomerase II DNA gyrase, and cytochrome P450 14 alpha-sterol demethylase. In conclusion, all the results indicated the great prospective of the novel cobalt complexes for some potential clinical applications in the future.

The crystal structure of olanzapine form III.

The antipsychotic drug olanzapine is well known for its complex polymorphism. Although widely investigated, the crystal structure of one of its anhydrous polymorphs, form III, is still unknown. Its appearance, always in concomitance with forms II and I, and the impossibility of isolating it from that mixture, have prevented its structure determination so far. The scenario has changed with the emerging field of 3D electron diffraction (3D ED) and its great advantages in the characterization of polyphasic mixtures of nanosized crystals. In this study, we show how the application of 3D ED allows the ab initio structure determination and dynamical refinement of this elusive crystal structure that remained unknown for more than 20 years. Olanzapine form III is monoclinic and shows a similar but shifted packing with respect to form II. It is remarkably different from the lowest-energy structures predicted by the energy-minimization algorithms of crystal structure prediction.

The Curious Case of A31P, a Topology-Switching Mutant of the Repressor of Primer Protein: A Molecular Dynamics Study of Its Folding and Misfolding.

The effect of mutations on protein structures is usually rather localized and minor. Finding a mutation that can single-handedly change the fold and/or topology of a protein structure is a rare exception. The A31P mutant of the homodimeric Repressor of primer (Rop) protein is one such exception: This single mutation ─and as demonstrated by two independent crystal structure determinations─ can convert the canonical (left-handed/all-antiparallel) 4-α-helical bundle of Rop to a new form (right-handed/mixed parallel and antiparallel bundle) displaying a previously unobserved "bisecting U" topology. The main problem with understanding the dramatic effect of this mutation on the folding of Rop is to understand its very existence: Most computational methods appear to agree that the mutation should have had no appreciable effect, with the majority of energy minimization methods and protein structure prediction protocols indicating that this mutation is fully consistent with the native Rop structure, requiring only a local and minor change at the mutation site. Here we use two long (10 μs each) molecular dynamics simulations to compare the stability and dynamics of the native Rop versus a hypothetical structure that is identical with the native Rop but is carrying this single Alanine31 to Proline mutation. Comparative analysis of the two trajectories convincingly shows that, in contrast to the indications from energy minimization ─but in agreement with the experimental data─, this hypothetical native-like A31P structure is unstable, with its turn regions almost completely unfolding, even under the relatively mild 320 K NpT simulations that we have used for this study. We discuss the implication of these findings for the folding of the A31P mutant, especially with respect to the proposed model of a double-funneled energy landscape.

Indium(III) complexes with lapachol: cytotoxic effects against human breast tumor cells and interactions with DNA.

Lapachol (2-hydroxy-3-(3-methylbut-2-en-1-yl)naphthalene-1,4-dione) is a 1,4-naphthoquinone-derived natural product that presents numerous bioactivities and was shown to have cytotoxic effects against several human tumor cells. Indium(III) complexes with a variety of ligands also exhibit antineoplastic activity. Indium(III) complexes [In(lap)Cl2].4H2O (1), [In(lap)2Cl(Et3N)] (2), [In(lap)3]·2H2O (3) [In(lap)(bipy)Cl2] bipy = 2,2'-bipyridine (4) and [In(lap)(phen)Cl2] phen = 1,10-phenanthroline (5) were obtained with 2-hydroxy-3-(3-methylbut-2-en-1-yl)naphthalene-1,4-dione (lapachol). Crystal structure determinations for (4) and (5) revealed that the indium(III) center is coordinated to two O atoms from lapachol, two N atoms from 1,10-phenanthroline or 2,2'-bipyridine, and two chloride anions, in a distorted octahedral geometry. Although both complexes (4) and (5) interacted with CT-DNA in vitro by an intercalative mode, only 5 exhibited cytotoxicity against MCF-7 and MDA-MB breast tumor cells. 1,10-phenanthroline and complex (5) presented cytotoxic effects against MCF-7 and MDA-MB cells, with complex (5) being threefold more active than 1,10-phenanthroline on MCF-7 cells. In addition, complex (5) significantly reduced the formation of MDA-MB-231 colonies in a clonogenicity assay. The foregoing results suggest that further studies on the cytotoxic effects and cellular targets of complex (5) are of utmost relevance.

In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.

Chirality Determination of Nanocrystals by Electron Crystallography.

Chirality is a common phenomenon in nature and plays an important role in the properties of matter. The rational synthesis of chiral compounds and exploration of their applications in various fields require an unambiguous determination of their handedness. However, in many cases, determinations of the chiral crystal structure and chiral morphology have been a challenging task due to the lack of proper characterization methods, especially for nanosized crystals. Therefore, it is crucial to develop novel and efficient characterization methods. Owing to the strong interactions between matter and electrons, electron crystallography has become a powerful tool for structural analysis of nanomaterials. In recent years, methods based on electron crystallography, such as high-resolution electron microscopy imaging and electron diffraction, have been developed to unravel the chirality of nanomaterials. This brings new opportunities to the design, synthesis, and applications of versatile chiral nanomaterials. In this perspective, we summarize the recent methodology developments and ongoing research of electron crystallography for chiral structure and morphology determination of nanocrystals, including inorganic and organic materials, as well as highlight the potential and further improvement of these methods in the future.

Crystal structure determination and Hirshfeld surface analysis of N-acetyl-N-3-meth-oxy-phenyl and N-(2,5-di-meth-oxy-phen-yl)-N-phenyl-sulfonyl derivatives of N-[1-(phenyl-sulfon-yl)-1H-indol-2-yl]methanamine.

Two new [1-(phenyl-sulfon-yl)-1H-indol-2-yl]methanamine derivatives, namely, N-(3-meth-oxy-phen-yl)-N-{[1-(phenyl-sulfon-yl)-1H-indol-2-yl]meth-yl}acetamide, C24H22N2O4S, (I), and N-(2,5-di-meth-oxy-phen-yl)-N-{[1-(phenyl-sulfon-yl)-1H-indol-2-yl]meth-yl}benzene-sulfonamide, C29H26N2O6S2, (II), reveal a nearly orthogonal orientation of their indole ring systems and sulfonyl-bound phenyl rings. The sulfonyl moieties adopt the anti-periplanar conformation. For both compounds, the crystal packing is dominated by C-H⋯O bonding [C⋯O = 3.312 (4)-3.788 (8) Å], with the structure of II exhibiting a larger number, but weaker bonds of this type. Slipped π-π inter-actions of anti-parallel indole systems are specific for I, whereas the structure of II delivers two kinds of C-H⋯π inter-actions at both axial sides of the indole moiety. These findings agree with the results of Hirshfeld surface analysis. The primary contributions to the surface areas are associated with the contacts involving H atoms. Although II manifests a larger fraction of the O⋯H/H⋯O contacts (25.8 versus 22.4%), most of them are relatively distal and agree with the corresponding van der Waals separations.

Multi-physical channels response in a novel layered perovskite: (trans-4-methylcyclohexylammonium)2[CuCl4]

With the continuous development of society, the demand for multi-physical channel responsive materials continues to increase, and it is urgent to develop multi-channel responsive materials with excellent properties. Here, in this work, we reported a novel two-dimensional (2D) organic–inorganic hybrid perovskite, (trans-4-methylcyclohexylammonium)2[CuCl4] (1), with responsive multi-physical channels. 1 experienced a structural phase transition with switchable thermochromism and dielectric response, which has great potential in the application of responsive materials. Under thermal stimulation, crystal 1 displays a reversible color variation between green and yellow. Crystal structure determination reveals that the distortion of inorganic layers plays a predominant role in the thermochromism during the phase transition process. This finding presents a compelling avenue for developing multi-physical channel responsive materials and contributes valuable insights to the exploration of two-dimensional lead-free perovskites for applications in intelligent window technology.

Powder x-ray diffraction analysis with machine learning for organic-semiconductor crystal-structure determination

The crystal structure of organic semiconductors is an important factor that dominates various electronic properties, including charge transport properties. However, compared with the crystal structures of inorganic semiconductors, those of organic semiconductors are difficult to determine by powder x-ray diffraction (PXRD) analysis. Our proposed machine-learning (neural-network) technique can determine the diffraction peaks buried in noise and make deconvolution of the overlapped peaks of organic semiconductors, resulting in crystal-structure determination by the Rietveld analysis. As a demonstration, we apply the method to a few high-mobility organic semiconductors and confirm that the method is potentially useful for analyzing the crystal structure of organic semiconductors. The present method is also expected to be applicable to the determination of complex crystal structures in addition to organic semiconductors.

Electrosynthesis of Unusual Nonfcc Palladium Hydride Nanoparticles.

Intercalation of hydrogen into the palladium atomic layers during the growth of Pd nanoparticles can lead to the synthesis of unique palladium hydride phases. Here, we discover an unusual nonfcc palladium hydride nanoparticle, a structure that is not face-centered cubic (fcc), formed through coreduction of water molecules and Pd ions in solution. Crystal structure determination based on atomic electron tomography points to potential triclinic unit cells, indicating the presence of more than one nonfcc phase, with some of those being a stack of loosened and distorted close-packed layer of atoms. The probability of finding the nonfcc phase in single-crystalline particles varies depending on the number and distribution of contact area with other particles. Roughly half of the isolated and one side-coalesced single-crystal particles exhibit a nonfcc structure, while fcc dominates multiple side-coalesced single crystals as well as polycrystal particles. These observations suggest a coalescence-induced phase transition from a nonfcc to a stable fcc structure, due to the metastable nature of the nonfcc phases. While hydrogen is proven to be a key component for the synthesis of the nonfcc structure, there was limited formation of the unusual phase in a H2 gas bubbling system. Thus, electrochemical pathways can be promising for the in situ creation and study of unique metastable nanomaterials.

Structural Basis for Evasion of New SARS-CoV-2 Variants from the Potent Virus-Neutralizing Nanobody Targeting the S-Protein Receptor-Binding Domain

COVID-19 has caused millions of deaths and many times more infections worldwide, emphasizing the unpreparedness of the global health system in the face of new infections and the key role for vaccines and therapeutics, including virus-neutralizing antibodies, in prevention and containment of the disease. Continuous evolution of the SARS-CoV-2 coronavirus has been causing its new variants to evade the action of the immune system, which highlighted the importance of detailed knowledge of the epitopes of already selected potent virus-neutralizing antibodies. A single-chain antibody (“nanobody”) targeting the SARS-CoV-2 receptor-binding domain (RBD), clone P2C5, had exhibited robust virus-neutralizing activity against all SARS-CoV-2 variants and, being a major component of the anti-COVID-19 formulation “GamCoviMab”, had successfully passed Phase I of clinical trials. However, after the emergence of the Delta and XBB variants, a decrease in the neutralizing activity of this nanobody was observed. Here we report on the successful crystal structure determination of the RBD:P2C5 complex at 3.1 Å, which revealed the intricate protein–protein interface, sterically occluding full ACE2 receptor binding by the P2C5-neutralized RBD. Moreover, the structure revealed the developed RBD:P2C5 interface centered around residues Leu452 and Phe490, thereby explaining the evasion of the Delta or Omicron XBB, but not Omicron B.1.1.529 variant, as a result of the single L452R or F490S mutations, respectively, from the action of P2C5. The structure obtained is expected to foster nanobody engineering in order to rescue neutralization activity and will facilitate epitope mapping for other neutralizing nanobodies by competition assays.

Synthesis, crystal structure and photophysical properties of a dinuclear MnII complex with 6-(di-ethyl-amino)-4-phenyl-2-(pyridin-2-yl)quinoline.

A new quinoline derivative, namely, 6-(di-ethyl-amino)-4-phenyl-2-(pyridin-2-yl)quinoline, C24H23N3 (QP), and its MnII complex aqua-1κO-di-μ-chlorido-1:2κ4 Cl:Cl-di-chlorido-1κCl,2κCl-bis-[6-(di-ethyl-amino)-4-phenyl-2-(pyridin-2-yl)quinoline]-1κ2 N 1,N 2;2κ2 N 1,N 2-dimanganese(II), [Mn2Cl4(C24H23N3)2(H2O)] (MnQP), were synthesized. Their compositions have been determined with ESI-MS, IR, and 1H NMR spectroscopy. The crystal-structure determination of MnQP revealed a dinuclear complex with a central four-membered Mn2Cl2 ring. Both MnII atoms bind to an additional Cl atom and to two N atoms of the QP ligand. One MnII atom expands its coordination sphere with an extra water mol-ecule, resulting in a distorted octa-hedral shape. The second MnII atom shows a distorted trigonal-bipyramidal shape. The UV-vis absorption and emission spectra of the examined compounds were studied. Furthermore, when investigating the aggregation-induced emission (AIE) properties, it was found that the fluorescent color changes from blue to green and eventually becomes yellow as the fraction of water in the THF/water mixture increases from 0% to 99%. In particular, these color and intensity changes are most pronounced at a water fraction of 60%. The crystal structure contains disordered solvent mol-ecules, which could not be modeled. The SQUEEZE procedure [Spek (2015 ▸). Acta Cryst. C71, 9-18] was used to obtain information on the type and qu-antity of solvent mol-ecules, which resulted in 44 electrons in a void volume of 274 Å3, corresponding to approximately 1.7 mol-ecules of ethanol in the unit cell. These ethanol mol-ecules are not considered in the given chemical formula and other crystal data.

Crystal structure and spectroscopic determination of the phase transitions in methylammonium- and formamidinium bismuth iodide perovskites

Understanding of the structural properties of hybrid organic–inorganic perovskites (HOIPs) and their behavior is crucial for their use as photovoltaics and for the design and assembly of solar cells. As part of this work, a detailed study was conducted to further understand bismuth iodide perovskites, with a specific focus on the phase transitions of methylammonium and formamidinium analogs. A detailed analysis of the temperature-dependent IR spectra was also performed in order to analyze the structural changes that occur. The presence of five phases in the methylammonium bismuth iodide (MABiI) and four phases in formamidinium bismuth iodide (FABiI) were determined. An additional confirmation of the reported results was obtained from the differential scanning calorimetry. The ambiguities concerning the crystal structure of FABiI were resolved based on the results by X-ray powder diffraction (XRPD).