Linking solid-state phenomena via energy differences in `archetype crystal structures'.

Categorization underlies understanding. Conceptualizing solid-state structures of organic molecules with `archetype crystal structures' bridges established categories of disorder, polymorphism and solid solutions and is herein extended to special position and high-Z' structures. The concept was developed in the context of disorder modelling [Dittrich, B. (2021). IUCrJ, 8, 305-318] and relies on adding quantum chemical energy differences between disorder components to other criteria as an explanation as to why disorder - and disappearing disorder - occurs in an average structure. Part of the concept is that disorder, as probed by diffraction, affects entire molecules, rather than just the parts of a molecule with differing conformations, and the finding that an R·T energy difference between disorder archetypes is usually not exceeded. An illustrative example combining disorder and special positions is the crystal structure of oestradiol hemihydrate analysed here, where its space-group/subgroup relationship is required to explain its disorder of hydrogen-bonded hydrogen atoms. In addition, we show how high-Z' structures can also be analysed energetically and understood via archetypes: high-Z' structures occur when an energy gain from combining different rather than overall alike conformations in a crystal significantly exceeds R·T, and this finding is discussed in the context of earlier explanations in the literature. Twinning is not related to archetype structures since it involves macroscopic domains of the same crystal structure. Archetype crystal structures are distinguished from crystal structure prediction trial structures in that an experimental reference structure is required for them. Categorization into archetype structures also has practical relevance, leading to a new practice of disorder modelling in experimental least-squares refinement alluded to in the above-mentioned publication.

Invariom refinement of a new monoclinic solvate of thiostrepton at 0.64†Šresolution.

A new monoclinic solvate containing two molecules of the thiopeptide antibiotic thiostrepton in the asymmetric unit has been crystallized in space group P2(1). Single-crystal diffraction data to a resolution of 0.64 Šwere collected at the SLS synchrotron, allowing structure solution by direct methods and resolution of the disorder present. Valence electron density can be observed in the Fourier residual density from refinement with the independent-atom model, which is a prerequisite for successful application of more sophisticated aspherical-atom scattering factors such as the invariom model when aiming to improve the structural model. Invariom refinement improves quality indicators such as R1(F) for thiostrepton, as previously demonstrated for small molecules. The nonspherical electron-density model also allows the direct derivation of a dipole moment and an electrostatic potential for the whole molecule, which is discussed in the context of antibiotic activity and molecular recognition.

Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15.

A subset of patients with Angelman and Prader-Willi syndrome have apparently normal chromosomes of biparental origin, but abnormal DNA methylation at several loci within chromosome 15q11-13, and probably have a defect in imprinting. Using probes from a newly established 160-kb contig including D15S63 (PW71) and SNRPN, we have identified inherited microdeletions in two AS families and three PWS families. The deletions probably affect a single genetic element that we term the 15q11-13 imprinting centre (IC). In our model, the IC regulates the chromatin structure, DNA methylation and gene expression in cis throughout 15q11-13. Mutations of the imprinting centre can be transmitted silently through the germline of one sex, but appear to block the resetting of the imprint in the germline of the opposite sex.

Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

Invariom modeling of ceftazidime pentahydrate: molecular properties from a 200 second synchrotron microcrystal experiment.

The structure of ceftazidime pentahydrate, a third generation cephalosporin antibiotic, is reported. Data collection was carried out in a remarkably short time with synchrotron radiation and the latest detector technology, illustrating that single-crystal X-ray diffraction can be used as a technique for screening hundreds of compounds in a short amount of time. Structure refinement made use of invarioms, namely non-spherical scattering factors, which allow more information to be derived from a diffraction experiment. Properties that can be screened are bond-topological parameters, empirical hydrogen-bond energies, molecular dipole moments and electrostatic potentials.

[Epithelioid angiomyolipoma of the kidney after successfully treated malignant melanoma]. / Epitheloides Angiomyolipom der Niere nach stattgehabtem malignem Melanom.

BACKGROUND: Therapy of epitheloid angiomyolipomas (eAML) may be challenging, since unlike classical angiomyolipomas this rare subclass of benign mesenchymal angiomyolipomas may present with lymph node metastases, local recurrent disease, and/or systemic metastatic disease in up to 30% of cases. OBJECTIVES: We report here for the first time in Germany a case of eAML after successful treatment of malignant melanoma. MATERIALS AND METHODS: Clinical and histological findings as well as results of the genetic analysis of the angiomyolipoma are presented. RESULTS: A somatic, truncating mutation of the TSC2 gene was found in the angiomyolipoma. CONCLUSION: The relationship to histologically similar tumor entities are presented and therapeutic options based on the genetic classification are discussed.

Validation of experimental charge densities: refinement of the macrolide antibiotic roxithromycin.

Multipole refinements of larger organic molecules have so far been limited to a few exceptional cases. We report an investigation of the detailed experimental electron-density distribution (EDD) of roxithromycin, a macrolide antibiotic consisting of 134 atoms. Although the experimental multipole refinement on high-resolution synchrotron data converged smoothly, validation of the electron density by calculation of an `experiment minus invariom' difference density revealed conformational disorder of the H atoms. Hydrogen disorder is shown to affect the EDD, the electrostatic potential and atomic properties as defined by Bader's quantum theory of atoms in molecules. A procedure to obtain the electron density distribution in the presence of disorder is proposed.

Charge-density study on cyclosporine A.

Two single-crystal X-ray diffraction data sets of cyclosporine A were measured to high resolution using synchrotron radiation at temperatures of 5 and 90 K. They allowed an accurate determination of its molecular and electronic structure. Three electron-density models based on pseudoatom scattering factors were compared in terms of derived bond topological properties and in terms of electron-density differences on a grid. In one model multipole parameters were freely refined, whereas in the other two models the density was built up from fixed database parameters from the invariom database and University at Buffalo Databank. The data quality not only allowed benchmarking of the quality of both databases with the refined density, but also judgement of the feasibility of a multipole refinement of a larger oligopeptide structure such as cyclosporine A. Both databases performed equally well and reproduced the experimentally determined charge density satisfactorily.

Polymer-based controlled-release fed-batch microtiter plate - diminishing the gap between early process development and production conditions.

BACKGROUND: Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. RESULTS: Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. CONCLUSIONS: The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible.

Polymer-based ammonium-limited fed-batch cultivation in shake flasks improves lipid productivity of the microalga Chlorella vulgaris.

The aim of this work was to study ammonium-limited fed-batch conditions in heterotrophic C. vulgaris shake flask cultivations. Therefore, an innovative polymer-based ammonium release technique (polymer beads) was developed. Using these beads in shake flasks, C. vulgaris cultivations resulted in simultaneous growth and lipid accumulation. Lipid productivity was increased by 43% compared to batch cultivations. Furthermore, by online monitoring of the metabolic activity (RAMOS technique), unlimited growth and depletion of nutrients could be identified. A previously unknown sulfur limitation was detected in the applied Bold's Basal Medium. Combining the ammonium release beads with the RAMOS technique proved to be an efficient method for microalgae process development.

Improvement of anisotropic displacement parameters from invariom-model refinements for three L-hydroxylysine structures.

Three L-hydroxylysine structures have been determined at 100 K by single-crystal X-ray diffraction. High-resolution data using either a laboratory or synchrotron source were collected and subjected to invariom- and independent atom-model (IAM) refinements. Anisotropic displacement parameters (ADPs) obtained from invariom refinement were compared (i) with results from a full multipole and (ii) with an IAM high-order refinement. Differences were visualized with the program PEANUT and were complemented by quantitative results from a Hirshfeld test. Influences of scale factor differences, and of refinement against F;2 versus F, have been investigated. Systematic errors were observed in the IAM, especially when only low-order data were available. Although these errors were reduced in high-order IAM refinements, they only disappeared in charge density--and likewise--invariom refinements.

Azulene revisited: solid-state structure, invariom modeling and lattice-energy minimization of a classical example of disorder.

The molecular and solid-state structure of azulene both raise fundamental questions. Therefore, the disordered crystal structure of azulene was re-refined with invariom non-spherical atomic scattering factors from new single-crystal X-ray diffraction data with a resolution of d = 0.45 Å. An unconstrained refinement results in a molecular geometry with Cs symmetry. Refinements constrained to fulfill C2v symmetry, as observed in the gas phase and in high-level ab initio calculations, lead to similar figures of merit and residual densities as unconstrained ones. Such models are consistent with the structures from microwave spectroscopy and electron diffraction, albeit they are not the same. It is shown that for the disorder present in azulene, the invariom model describes valence electron density as successfully as it does for non-disordered structures, although the disorder still leads to high correlations mainly between positional parameters. Lattice-energy minimizations on a variety of ordered model structures using dispersion-corrected DFT calculations reveal that the local deviations from the average structure are small. Despite the molecular dipole moment there is no significant molecular ordering in any spatial direction. A superposition of all ordered model structures leads to a calculated average structure, which explains not only the experimental determined atomic coordinates, but also the apparently unusual experimental anisotropic displacement parameters.

Can the interaction density be measured? The example of the non-standard amino acid sarcosine.

The experimental charge density rho(r) of the non-standard amino acid sarcosine has been determined based on an extensive and complete data set measured at 100 K to high resolution (sin theta/lambda = 1.18 A(-1)) by single-crystal X-ray diffraction. Anisotropic thermal motion of the H atoms, obtained from TLS + ONIOM cluster methods, was included in the structural model. Based on the multipole-model geometry, the theoretical Hartree-Fock interaction density of a molecule in the crystal has been calculated with CRYSTAL98. It manifests itself in local rearrangements of rho(r) and can be reproduced with a multipole projection via simulated structure factors. An attempt has also been made to obtain the interaction density from a combination of experimental and theoretical charge densities using either a whole-molecular calculation or the invariom database. Agreement with the periodic Hartree-Fock interaction density is qualitative. It is shown that invarioms reproduce the features of the theoretical multipole-projected whole-molecular electron density, and can be used to approximate it.

Invarioms for improved absolute structure determination of light-atom crystal structures.

The determination of molecular absolute configuration from an X-ray analysis for structures that contain only light elements is challenging owing to the weak anomalous dispersion signal. The achievable precision of the Flack x parameter for such structures is therefore limited, especially when the independent-atom model is employed. Invariom modelling can improve this situation. Invarioms are theoretically predicted pseudoatoms within the Hansen & Coppens multipole formalism. They are transferable from one molecule to another and provide generalized aspherical atomic form factors. It is shown that, by application of the invariom approach, the precision and standard uncertainty of the Flack x parameter and therefore the reliability of deducing molecular chirality in an absolute structure determination can be improved.

Oxidative stress related markers in the "VITA" and the centenarian projects.

Oxidative stress seems to play an important role in the pathophysiology of Alzheimer's disease (AD). At present there are no easily accessible biochemical markers for AD. We performed activity assays for platelet MAO-B and erythrocyte Cu/Zn-SOD as well as Western blotting for these two proteins. Moreover, we assessed plasma lactoferrin and performed RFLP-analysis for the MAO-B-intron-13-polymorphism in patients from the Vienna-Transdanube Aging (VITA) and from the so called centenarian project. The first one, VITA, is a community-based cohort study of all 75 years old inhabitants of a geographical region of Vienna. The centenarian project investigates chronic care in-old patients suffering from AD. In both sexes platelet MAO-B activity increased significantly in the AD group, and Cu/Zn-SOD activity decreased, but the latter effect was significant only in females. No significant difference was found regarding plasma lactoferrin. No correlation was found between MAO-Bi13 and MAO-B platelet activity or allele MAO-Bi13 and disease frequency. These results point to the possibility that a combination of MAO-B and SOD activity levels might be useful tools for an early diagnosis of AD.

The invariom model and its application: refinement of D,L-serine at different temperatures and resolution.

Three X-ray data sets of the same D,L-serine crystal were measured at temperatures of 298, 100 and 20 K. These data were then evaluated using invarioms and the Hansen & Coppens aspherical-atom model. Multipole populations for invarioms, which are pseudoatoms that remain approximately invariant in an intermolecular transfer, were theoretically predicted using different density functional theorem (DFT) basis sets. The invariom parameters were kept fixed and positional and thermal parameters were refined to compare the fitting against the multi-temperature data at different resolutions. The deconvolution of thermal motion and electron density with respect to data resolution was studied by application of the Hirshfeld test. Above a resolution of sin theta/lambda approximately 0.55 A-1, or d approximately 0.9 A, this test was fulfilled. When the Hirshfeld test is fulfilled, a successful modeling of the aspherical electron density with invarioms is achieved, which was proven by Fourier methods. Molecular geometry improves, especially for H atoms, when using the invariom method compared to the independent-atom model, as a comparison with neutron data shows. Based on this example, the general applicability of the invariom concept to organic molecules is proven and the aspherical density modeling of a larger biomacromolecule is within reach.

Predictive QSAR models for estimating ecotoxic hazard of plant-protecting agents: target and non-target toxicity.

Quantitative structure-activity relationships (QSARs) are evaluated for predicting pesticides' effects on non-target and target organisms: aquatic biota, mammals, soil micro-organisms, and plants. Satisfactory estimates of pesticides' fish toxicity are obtained from log POW-dependent QSARs derived using chemicals of similar polarity and reactivity. Algal toxicity of herbicidal compounds reflects interactions with the electron transport chain in photosystem II and can be modelled by QSARs describing the Hill reaction inhibition. The ranking in effects on soil micro-organisms is evaluated from models derived using in-vitro bacteria systems. Mammalian toxicity can be estimated by QSARs using the partition coefficient log POW and electronic terms derived by MNDO quantum mechanical calculations. In general, the targets are more susceptible than the non-target species towards phenylurea herbicides. Plants and algae constitute the most sensitive populations, corresponding to the same mode of action. Differences in mode of action towards bacteria, rats, and fish, which are similarly sensitive organisms, are revealed by QSAR analyses.

The generalized invariom database (GID).

Invarioms are aspherical atomic scattering factors that enable structure refinement of more accurate and more precise geometries than refinements with the conventional independent atom model (IAM). The use of single-crystal X-ray diffraction data of a resolution better than sin θ/λ = 0.6 Å(-1) (or d = 0.83 Å) is recommended. The invariom scattering-factor database contains transferable pseudoatom parameters of the Hansen-Coppens multipole model and associated local atomic coordinate systems. Parameters were derived from geometry optimizations of suitable model compounds, whose IUPAC names are also contained in the database. Correct scattering-factor assignment and orientation reproduces molecular electron density to a good approximation. Molecular properties can hence be derived directly from the electron-density model. Coverage of chemical environments in the invariom database has been extended from the original amino acids, proteins and nucleic acid structures to many other environments encountered in organic chemistry. With over 2750 entries it now covers a wide sample of general organic chemistry involving the elements H, C, N and O, and to a lesser extent F, Si, S, P and Cl. With respect to the earlier version of the database, the main modification concerns scattering-factor notation. Modifications improve ease of use and success rates of automatic geometry-based scattering-factor assignment, especially in condensed hetero-aromatic ring systems, making the approach well suited to replace the IAM for structures of organic molecules.

On QM/MM and MO/MO cluster calculations of all-atom anisotropic displacement parameters for molecules in crystal structures.

The practical aspects of ab initio calculation of anisotropic displacement parameters (ADPs) for molecules in crystal structures are investigated. Computationally efficient approaches to calculate ADPs are QM/MM or MO/MO methods, where quantum chemical calculations are split into a high-level and a low-level part. Such calculations allow geometry optimizations and subsequent frequency calculations of a central molecule in a cluster of surrounding molecules as found in the crystal lattice. The frequencies and associated displacements are then converted into ADPs. A series of such calculations were performed with different quantum chemical methods and basis sets on the three zwitterionic amino-acid structures of L-alanine, L-cysteine and L-threonine, where high-quality low-temperature X-ray data are available. To scale and compare calculated ADPs, X-ray ADPs from invariom refinement were used. The future use of calculated ADPs will include the investigation of systematic errors in experimental X-ray diffraction data. Completion of an isotropic structural model is already possible. Calculated ADPs might also make it possible to perform charge-density studies on data sets of limited resolution/coverage as obtained from weak scatterers, high-pressure measurements or to deconvolute electron density obtained from the maximum-entropy method.