Crystallographic Approaches Research Articles

Covalent Probes To Capture Legionella pneumophila Dup Effector Enzymes.

Upon infection of host cells, Legionella pneumophila releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein. We installed reactive warheads on chemically prepared ribosylated ubiquitin to generate a set of probes targeting these Legionella enzymes. In vitro tests on recombinant DupA revealed that a vinyl sulfonate warhead was most efficient in covalent complex formation. Mutagenesis and X-ray crystallography approaches were used to identify the site of covalent cross-linking to be an allosteric cysteine residue. The subsequent application of this probe highlights the potential to selectively enrich the Dup enzymes from Legionella-infected cell lysates.

Dynamic and structural insights into allosteric regulation on MKP5 a dual-specificity phosphatase.

Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK.

Evaluation of charge-assisted hydrogen bonds and weak intermolecular interaction in trimethoprim 2-aminobenzoate: A combined crystallographic and theoretical approach

A comprehensive investigation of the weak non-covalent interactions within the crystal structure of trimethoprim 2-aminobenzoate (2,6-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidin-1-ium 2-aminobenzoate) is presented. The protonation of trimethoprim and the deprotonation of 2-aminobenzoate were confirmed by NMR spectroscopy and a single-crystal X-ray diffraction study. Qualitative Hirshfeld surface analysis identified intermolecular H···H, O···H, and C···H contacts as the three primary interactions within the title salt. Fourteen molecular pairs (M1AA−M14AB) were extracted from the crystal packing of the title salt using the PIXEL method. These molecular dimers were further characterized using the QTAIM and DFT approaches. Seven intermolecular pairs (M1AA−M7BB) formed between like-charged species (i.e. +1···+1 or -1···-1) resulted in destabilization, with total energies (Etot) ranging from 67.2 to 200.1 kJ mol-1. Conversely, molecular pairs formed between oppositely charged species (i.e. +1···-1) exhibited stabilizing behavior, with interaction energies ranging from -133.7 to -449.7 kJ mol-1. Topological analysis of charge density revealed that two N–H···O hydrogen bonds between cation and anion displayed an intermediate character between shared and closed-shell interactions. Additionally, comparison with previously reported similar TMP salts (CSD ref. code: CESRUN, HURMOW and PARWUB) demonstrated 3D-isostructural packing features in the crystal structure of the title salt.

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.

Spectroscopically Validated pH-dependent MSOX Movies Provide Detailed Mechanism of Copper Nitrite Reductases

Copper nitrite reductases (CuNiRs) exhibit a strong pH dependence of their catalytic activity. Structural movies can be obtained by serially recording multiple structures (frames) from the same spot of a crystal using the MSOX serial crystallography approach. This method has been combined with on-line single crystal optical spectroscopy to capture the pH-dependent structural changes that accompany during turnover of CuNiRs from two Rhizobia species. The structural movies, initiated by the redox activation of a type-1 copper site (T1Cu) via X-ray generated photoelectrons, have been obtained for the substrate-free and substrate-bound states at low (high enzymatic activity) and high (low enzymatic activity) pH. At low pH, formation of the product nitric oxide (NO) is complete at the catalytic type-2 copper site (T2Cu) after a dose of 3 MGy (frame 5) with full bleaching of the T1Cu ligand-to-metal charge transfer (LMCT) 455 nm band (S(σ)Cys → T1Cu2+) which in itself indicates the electronic route of proton-coupled electron transfer (PCET) from T1Cu to T2Cu. In contrast at high pH, the changes in optical spectra are relatively small and the formation of NO is only observed in later frames (frame 15 in Br2DNiR, 10 MGy), consistent with the loss of PCET required for catalysis. This is accompanied by decarboxylation of the catalytic AspCAT residue, with CO2 trapped in the catalytic pocket.

Structure-based mapping of the histone-binding pocket of KDM4D using functionalized tetrazole and pyridine core compounds

KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.

A crystallographic approach to symmetry-breaking in fluid layers

A crystallographic approach to symmetry-breaking in fluid layers

Planting the Seeds of a Decision Tree for Ionic Liquids: Steric and Electronic Impacts on Melting Points of Triarylphosponium Ionic Liquids.

While machine learning and artificial intelligence offer promising avenues in the computer-aided design of materials, the complexity of these computational techniques remains a barrier for scientists outside of the specific fields of study. Leveraging decision tree models, inspired by empirical methodologies, offers a pragmatic solution to the knowledge barrier presented by artificial intelligence (AI). Herein, we present a model allowing for the qualitative prediction of melting points of ionic liquids derived from the crystallographic analysis of a series of phosphonium-based ionic liquids. By carefully tailoring the steric and electronic properties of the cations within these salts, trends in the melting points are observed, pointing toward the critical importance of π interactions to forming the solid state. Quantification of the percentage of these π interactions using modern quantum crystallographic approaches reveals a linear trend in the relationship of C-Hπ and π-π stacking interactions with melting points. These structure-property relationships are further examined by using computational studies, helping to demonstrate the inverse relationship of dipole moments and melting points for ionic liquids. The results provide valuable insights into the features and relationships that are consistent with achieving low Tm values in phosphonium salts, which were not apparent in earlier studies. The data gathered are presented in a simple decision tree format, allowing for visualization of the data and providing guidance toward developing yet unreported compounds.

NIST Workshop: Integrating Crystallographic and Computational Approaches to Carbon-Capture Materials for the Mitigation of Climate Change (October 31–November 1, 2023)

The NIST Workshop: Integrating Crystallographic and Computational Approaches to Carbon-Capture Materials for the Mitigation of Climate Change took place from October 31–November 1, 2023 at the National Cybersecurity Center of Excellence (NCCoE) Compound in Rockville, MD, which is an off-campus NIST facility. This workshop provided a forum for experimentalists and theorists working on the structural aspects of CO2 capture and sequestration materials to review the current state of the art in this field and discuss opportunities for collaborative research required to develop tools for rapid determination of the structure and its effect on the direct air capture performance in porous solid sorbents. We had a total of 33 international participants (18 invited speakers) from 17 institutions who were experimentalists and theorists from academia, government, and industry. The workshop was a great success.

Unveiling structural features, chemical reactivity, and bioactivity of a newly synthesized purine derivative through crystallography and computational approaches

We introduce the synthesis and characterization of a novel purine derivative, 2-amino-6‑chloro-N,N-diphenyl-7H-purine-7-carboxamide. X-ray crystallography was utilized to elucidate its molecular and crystal structure. A comprehensive crystal packing analysis uncovered a network of diverse intermolecular interactions, including classical and unconventional hydrogen bonding. Remarkably, a unique halogen···π (C—Cl···π(ring)) interaction was identified and theoretically analyzed within a multi-approach quantum mechanics (QM) framework, revealing its lone-pair⋯π (n→π*) nature. Furthermore, insights into the electronic and chemical reactivity properties are provided by means of Conceptual Density Functional Theory (CDFT) at wB97X-D/aug-cc-pVTZ level. The compound's drug-likeness, pharmacokinetics, and toxicology profiles are assessed using ADMETlab 2.0. Finally, molecular docking simulations were conducted to evaluate its bioactivity as a potential cyclooxygenase-2 (COX-2) inhibitor. This study significantly advances our understanding of purine structure and reactivity, offering valuable insights for the development of targeted purine-based COX-2 inhibitors and anticancer therapeutics.

Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Elastic and piezoelectric properties of β-glycine - a quantum crystallography view on intermolecular interactions and a high-pressure phase transition.

The effect of hydrostatic compression on the elastic and electronic properties of β-glycine was studied using a quantum crystallography approach. The interrelations between the changes in the microscopic quantum pressure in the electronic continuum, macroscopic compressibility and piezoelectricity were considered. The geometries and energies of hydrogen bonds in the crystal structure of β-glycine were considered as functions of pressure before and after a phase transition into the β'-phase in relation to the mechanism of this phase transition.

Integrating crystallographic and computational approaches to carbon-capture materials for the mitigation of climate change

This article presents a perspective on the state of the art in the structure determination of microporous carbon-capture materials and paths toward future progress in this field, as discussed the NIST workshop of the same title.

Magnesium cations as templates for the self-assembly of supramolecular luminescent {Mg@[B18φ34-35]}-clusters.

Solvothermal reaction of magnesium nitrate and boron oxide in N,N-dimethylformamide produced a number of particularly complex supramolecular magnesium borates. Five topologically different types of negatively charged {Mg@[B18φ34-35]}-clusters, φ = O, OH, were observed with the magnesium cation as a core and octadecaborate anions as shells. The clusters assemble via common borate polyhedra forming 1D chains, a 2D mesoporous layer, and 3D mesoporous frameworks with an effective channel width of up to 16 Å. Topological analysis of the clusters in combination with the modular crystallography approach indicates that numerous new functional materials can be obtained by varying their assembly mode. At least one compound containing such clusters exhibits a very strong luminescence.

Probing assembly/disassembly of ordered molecular hydrogels.

Supramolecular hydrogels have a wide range of applications in the biomedical field, acting as scaffolds for cell culture, matrices for tissue engineering and vehicles for drug delivery. L-Phenylalanine (Phe) is a natural amino acid that plays a significant role in several physiological and pathophysiological processes (phenylketonuria and assembly of fibrils linked to tissue damage). Since Myerson et al. [Chem. Eng. Commun., 2002, 189(8), 1079-1090] reported that Phe forms a fibrous network in vitro, Phe's self-assembly processes in water have been thoroughly investigated. We have reported structural control over gelation by introduction of a halogen atom in the aromatic ring of Phe, driving changes in the packing motifs, and therefore, dictating gelation functionality. The additional level of control gained over supramolecular gelation via the preparation of multi-component gel systems offers significant advantages in tuning functional properties of such materials. Gaining molecular-level information on the distribution of gelators between the inherent structural and dynamic heterogeneities of these materials remains a considerable challenge. Using multicomponent gels based on Phe and amino-L-phenylalanine (NH2-Phe), we will explore the patterns of ordered/disordered domains in the gel fibres and will attempt to come up with general trends of interactions in the gel fibres and at the fibre/solution interfaces. Phe and NH2-Phe were found to self-assemble in water into crystalline hydrogels. The determined faster dynamics of exchange between the gel and solution states of NH2-Phe in comparison with Phe were correlated with weaker intermolecular interactions, highlighting the role of head groups in dictating the strength of intermolecular interactions. In the mixed Phe/NH2-Phe systems, at a low concentration of NH2-Phe, disruption of the network was promoted by interference of the aliphatics of NH2-Phe with the electrostatic interactions between Phe molecules. At high concentrations of NH2-Phe, multiple-gelator hydrogels were formed with crystal habits different from those of the pure gel fibres. NMR crystallography approaches combining the strengths of solid- and solution-state NMR proved particularly suitable to obtain structural and dynamic insights into the "ordered" fibres, solution phase and fibre/solution interfaces in these gels. These findings are supported by a plethora of experimental (diffraction, rheology, microscopy and thermal analysis) and computational methods.

Identification of fragments targeting SMYD3 using highly sensitive kinetic and multiplexed biosensor-based screening.

A 1056-membered fragment library has been screened against SMYD3 using a novel multiplexed experimental design implemented in a grating coupled interferometry (GCI)-based biosensor. SMYD3 is a prospective target for anticancer drugs and the focus has initially been on discovery of inhibitors of its lysine methyl transferase activity. However, it has multiple protein interaction partners and several potential roles in carcinogenesis. It therefore remains unclear what mode of action ligands targeting the protein should have. Our goal was therefore to identify new ligands and discriminate hits that interact with the active site and those that interact with other sites. In addition, we were interested in selecting hits based on kinetic features rather than affinity. Screening was done in parallel against SMYD3 alone or SMYD3 with the active site blocked by a tight binding inhibitor. Hit selection was primarily based on dissociation rates. In total, 20 fragments were selected as hits, of which half apparently targeted the active site and half targeted other sites. Twelve of the hits were selected for structural analysis using X-ray crystallography in order to identify binding sites and modes of binding. Four of the hits were successfully identified in crystal structures with SMYD3; the others did not show any electron densities for ligands in the crystals. Although it might be possible to optimize the crystallography approach for a better success rate, it was clear that the sensitivity and time resolution of the biosensor assay was exceptional and enabled kinetic rate constants to be estimated for fragments. Fragments are typically considered to interact too rapidly for such quantification to be possible. This approach consequently represents a paradigm shift. In addition, the multiplexed approach allows ligands targeting different sites to be rationally selected already in the fragment library screening stage.

Structural complexity of γ-Al2O3: The nature of vacancy ordering and the structure of complex antiphase boundaries

The structure of γ-Al2O3 remains undetermined despite decades of research. This is due to the high degree of disorder, which poses significant challenges for structural analysis using conventional crystallographic approaches. Herein, we study the structure of γ-Al2O3 with scanning transmission electron microscopy (STEM) and ab-initio calculations and show that the structure can be interpreted as a domain microstructure of defective spinel interconnected via non-spinel segments that form the basis of antiphase boundaries (APBs). The spinel domains have a distinctive preference for vacancy ordering, which can be rationalized in terms of a structure with a stacking disorder. Tetragonal P41212 or monoclinic P21 models, all based on an identical motif but with a different stacking sequence, can be considered as representative ordered forms. Individual spinel domains are interconnected via distinct non-spinel bonding environment of δ-Al2O3, which we assign as complex APBs. The most common cAPB consists of a single delta motif with thickness of just 0.6 nm on (001), with the resulting displacement a/4 [101]. Remarkably, the cAPBs are shown to energetically stabilize the spinel domains of γ-Al2O3 explaining their high abundance. We demonstrate how the tetragonal distortions naturally arise in this intricate microstructure and place the proposed model in the context of phase transformations to high-temperature transition aluminas.

PDZome-wide and structural characterization of the PDZ-binding motif of VANGL2

VANGL2 is a core component of the non-canonical Wnt/Planar Cell Polarity signaling pathway that uses its highly conserved carboxy-terminal type 1 PDZ-binding motif (PBM) to bind a variety of PDZ proteins. In this study, we characterize and quantitatively assess the largest VANGL2 PDZome-binding profile documented so far, using orthogonal methods. The results of our holdup approach support VANGL2 interactions with a large panel of both long-recognized and unprecedented PDZ domains. Truncation and point mutation analyses of the VANGL2 PBM establish that, beyond the strict requirement of the P-0 / V521 and P-2 / T519 amino acids, upstream residues, including E518, Q516 and R514 at, respectively, P-3, P-5 and P-7 further contribute to the robustness of VANGL2 interactions with two distinct PDZ domains, SNX27 and SCRIBBLE-PDZ3. In agreement with these data, incremental amino-terminal deletions of the VANGL2 PBM causes its overall affinity to progressively decline. Moreover, the holdup data establish that the PDZome binding repertoire of VANGL2 starts to diverge significantly with the truncation of E518. A structural analysis of the SYNJ2BP-PDZ/VANGL2 interaction with truncated PBMs identifies a major conformational change in the binding direction of the PBM peptide after the P-2 position. Finally, we report that the PDZome binding profile of VANGL2 is dramatically rearranged upon phosphorylation of S517, T519 and S520. Our crystallographic approach illustrates how SYNJ2BP accommodates a S520-phosphorylated PBM peptide through the ideal positioning of two basic residues, K48 and R86. Altogether our data provides a comprehensive view of the VANGL2 PDZ network and how this network specifically responds to the post-translation modification of distinct PBM residues. These findings should prove useful in guiding future functional and molecular studies of the key PCP component VANGL2.

Tunable d33/d33* for MPB (Ba0.85Ca0.15) (Zr0.1Ti0.9)O3 lead-free piezoceramic by crystallographic texturing approach

The morphotropic phase boundary (MPB) Ba0.85Ca0.15Zr0.10Ti0.90O3 composition is successfully transferred into <001>-textured ceramic having Lotgering factor f-74 % (abbreviated as BCZT-T74) using high aspect ratio BaTiO3-template particles via templated grain growth (TGG) method. The microstructural aspects reveal the distinctive grain growth formation for non-textured (BCZT-NT) and textured (BCZT-T74) ceramics; confirming the proper grain growth formation for textured ceramics. The polarization behavior was tested at 1 Hz, 5 Hz, and 10 Hz electric field frequencies by applying a voltage bias of 1000 V; the BCZT-NT ceramics show more variation in remnant polarization (Pr) as well as maximum polarization (Pmax) peak position while BCZT-T74 ceramics exhibit nearly steady values. The frequency-dependent variation in Pr (i.e. ΔPr) for non-textured and textured BCZT ceramics is ±11.08 % and ±3.25 % respectively in the frequency range 1Hz–10Hz at an applied voltage bias of 1000 V. The bipolar strain-electric field (S-E) butterfly loop evidences the typical piezoelectric nature; for both non-textured and textured BCZT ceramics having the effective piezoelectric strain coefficient d33* of 576 pm/V and 657 pm/V respectively. The BCZT-NT and BCZT-T74 ceramics revealed the average positive strain % values of 0.063 % and 0.1123 % and negative strain % values of 0.003985 % and 0.02281 % respectively at the applied voltage 1000 V functioning at 1Hz. The enhancement in negative strain% for BCZT-T74 ceramics is > 5.70 times higher than BCZT-NT ceramics. The variation in positive strain (i.e. ΔS+ve) and variation in negative strain (i.e. ΔS-ve) for non-textured ceramics is ±11.74 % and ±67.34 %, however, for textured ceramics is only ±3.33 % and ±5.00 % respectively. The BCZT-NT and BCZT-T74 ceramics exhibit the piezoelectric charge coefficient (d33) of 370 pC/N and 520 pC/N (1.4 times that of BCZT-NT ceramics) respectively. The electrostrictive coefficient (Q33) of BCZT-T74 ceramics is 0.039 m4/C2, which is > 2 times higher than BCZT-NT ceramics as well as commercially used toxic PZT-5H ceramics. Therefore, less variation in a strain, as well as polarization nature with varying frequencies and enhancement in piezo properties, suggests the present textured MPB BCZT-T74 ceramics is a promising candidate for piezoelectric devices functioning at the wide range of applied electric field frequencies.

Regioselective synthesis, structural and bioactivity of acetylated 1,5-benzodiazepin-2-one derivatives — X-ray crystallography, DFT and molecular docking approaches

Benzodiazepines, a well-studied class of seven-membered aromatic heterocycles containing two nitrogen atoms, have shown great promise as therapeutic agents. Efficient protocols have been developed to synthesize a variety of functionalized benzodiazepines. This study focuses on the synthesis of three derivatives of 1,5-benzodiazepin-2-one through regioselective acetylation using acetyl chloride under various conditions. One of the derivatives was characterized for its molecular and crystal structure using X-ray crystallography, Hirshfeld surfaces, and density functional theory computations (at wB97X-D/aug-cc-pVTZ level). The synthesized compounds were then subjected to in vitro evaluation to assess their antibacterial and antifungal activities against multiple strains. Additionally, their antioxidant and anti-inflammatory properties were examined. The results showed different levels of inhibitory effects on the growth of microorganisms, with all compounds displaying moderate to significant biological activities. Furthermore, molecular docking simulations were conducted to investigate the binding interactions of the compounds with bacterial and fungal targets. This research enhances our understanding of the chemistry of benzodiazepine derivatives and further highlights their therapeutic potential.