Entwicklung theoretischer und experimenteller Methoden für die zeitaufgelöste Laue-Protein-Kristallographie

New theoretical, experimental and computational methods for time-resolved Laue crystallography of macromolecules have been developed. A solution to the energy-overlap problem in Laue diffraction is described that does not require redundancy in the measurements. The new method follows a Bayesian approach with multi-dimensional probability density functions. The intensity components of reflection multiplets are deconvoluted, and estimates of their precision are obtained. The Laue patterns are processed to their physically relevant wavelength-dependent resolution limit; no "soft-parameters" are involved. The power of the method is demonstrated by the test applications to bovine trypsin, sperm whale myoglobin and 2Mn-catalase from Thermus thermophilus. In the example of trypsin, the completeness at low and medium resolution as well as at very high resolution (1.4 e) is enhanced very substantially as compared to standard procedures; the "low-resolution hole" problem is practically solved. As a consequence, the contrast in electron density maps improves so far that they become comparable in quality to maps from monochromatic data of the same resolution. The new method is of interest for all types of Laue diffraction experiments, in particular for single-shot time resolved studies on short time scales. In a stroboscopic test study of ground-state CO myoglobin, the Bayesian method permitted to obtain the electron density maps that showed the correct ligand conformation at resolution 1.7 e. The resolution that was achieved using DORIS and the map contrast are higher as compared to the earlier experiments of this type that has been carried out at the third generation synchrotron source. The possibility to obtain interpretable maps in a single-shot Laue experiment is demonstrated for the example of cubic catalase. A limited-bandwidth Laue method using a graphite monochromator with 2.5% bandwidth has been developed and tested in study of lysozyme at ultra-high resolution (200 e are presented.Neue theoretische, experimentelle und rechnerische Methoden für die zeitaufgelöste Laue-Kristallographie von Makromolekülen wurden entwickelt. Eine Lösung für das Energieüberlappungsproblem der Laue-Diffraktion wird beschrieben, die keine Redundanz in den Messdaten erfordert. Die neue Methode nutzt Bayes-Statistik mit mehrdimensionalen Wahrscheinlichkeitsdichtefunktionen. Die Intensitätskomponenten der Reflexmultiplets werden entfaltet und ihre Genauigkeit abgeschätzt. Die Laue-Beugungsbilder werden bis zur jeweiligen physikalisch relevanten wellenlängenabhängigen Auflösungsgrenze bearbeitet, ohne dabei "Soft parameter" anzunehmen. Die Leistungsfähigkeit der Methode wird anhand von Testanwendungen an Rindertrypsin, Pottwal-Myoglobin und 2Mn-Katalase des Thermus thermophilus demonstriert. Im Fall von Trypsin liegt die Vollständigkeit bei niedrigen und mittleren sowie bei sehr hohen Auflösungen (1.4 e) im Vergleich zu Laue-Standardmethoden um ein Vielfaches höher. Das Problem der geringen Vollständigkeit im Breich niederer Auflösung ("low-resolution-hole") ist praktisch gelöst. Als Folge erhöht sich der Kontrast der Elektronendichtekarten so weit, dass sie in ihrer Qualität mit Karten vergleichbar sind, die auf monochromatischen Daten gleicher Auflösung beruhen. Die neue Methode ist für alle Arten von Laue-Beugungsexperimenten, insbesonders auch für zeitaufgelöste Studien aufgrund einer einzigen Aufnahme (single shot) in kurzem Zeitmaßstab von Interesse. In einer stroboskopischen Teststudie an CO-Myoglobin im Grundzustand ermöglichte es die Bayes-Methode, Elektronendichtekarten zu erhalten, die die korrekte Konformation der Liganden bei einer Auflösung von 1.7e zeigten. Die Auflösung und der Kontrast der Dichtekarten, die aufgrund von Messungen an DORIS erhalten wurden, sind höher als bei früheren Experimenten dieser Art, obwohl diese an Strahlungsquellen der dritten Generation durchgeführt wurden. Die Möglichkeit interpretierbare Karten mit Single-Shot-Laue-Experimenten zu erhalten, wird am Beispiel von kubischer Katalase demonstriert. Zudem wurde eine Laue-Methode mit reduzierter Bandbreite (2.5%) unter Verwendung eines Graphit-Monochromators entwickelt und am Beispiel von Lysozym bei extrem hoher Auflösung (< 1.2 e) getestet. Schließlich wurde ein neuer Algorithmus zur Autoindizierung von Laue-Beugungsmustern entwickelt und in einem Computerprogramm implementiert. Erfolgreiche Anwendungen dieses Verfahrens auf Strukturen mit vergleichsweise großen Zellkonstanten werden vorgestellt

Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima

Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding (Delta G). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar Delta G. However, the interaction with the CAA sequence was driven by change in enthalpy (Delta H), whereas the TCG site was bound with similar affinity due to smaller loss of entropy (Delta S). This thermodynamic mechanism that leads to at least two local optima likely affects many macromolecular interactions, as Delta G depends on two partially independent variables Delta H and Delta S according to the central equation of thermodynamics, Delta G = Delta H - T Delta S.Peer reviewe

Structure of a Sixteen Heme Cytochrome by X-Ray Crystallography.

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Experimental procedure for the characterization of radiation damage in macromolecular crystals

A novel automatic procedure to determine the sensitivity of macromolecular crystals to radiation damage is presented. The information extracted from this procedure can be directly used for optimal planning of data collection or/and beamline calibration

Carrying out an optimal experiment

Diffraction data collection parameters leading to optimal data quality are discussed in the context of different applications of these data

The safety and tolerability study of tiozonid in single dose with it increasing

A study of the safety and tolerability tiozonid manufacturing by JSC «Pharm-Sintez» (Russia) at the single dose with it increasing (25, 200, 400 and 600 mg) in healthy volunteers. In each group, the volunteers were enrolled sequentially, based on the interim evaluation of safety parameters. This study was conducted in compliance with the requirements of legislation and the ethical principles set forth in the Federal Law «On Circulation of Medicines» (number 61-FZ of April 12, 2010), the Declaration of Helsinki of the World Medical Association (1964, with subsequent amendments), GOST R 52379-2005 «Good clinical practice» The study drug tiozonid manufacturing by JSC «Pharm-Sintez» (Russia) has shown a sufficiently high safety and well-tolerated at single oral doses in the range of 25-600 mg

Revision of the genus Robbea (Stilbonematinae: Desmodoridae), worldwide abundant marine nematodes with chromophoric Fe-Br inclusions and the description of a new stilbonematine genus

Nematodes are one of the most widespread and abundant animal taxa across aquatic and terrestrial environments. In marine shallow-water, porous sediments, members of the subfamily Stilbonematinae may be found in high numbers. Stilbonematinae are characterized by their coat of symbiotic bacteria, which give the nematodes a white appearance, while the nematodes themselves are usually colourless. We identified several species of the genus Robbea (Desmodoridae: Stilbonematinae) in which live specimens had a conspicuous dark purple coloration of the glandular sense organs (GSOs), highly specialized epidermal glands that are prominent in the Stilbonematinae. The dark inclusions in the GSOs of Robbea contained high concentrations of iron and bromide, regardless of their habitat. Morphological and phylogenetic analyses show that the genus Robbea is paraphyletic and we define the new genus Cyathorobbea gen. nov. for species with sucker-shaped post-pharyngeal supplements in males. Furthermore, we describe five new Robbea species from around the world. Some Robbea species are morphologically very similar and required the use of principal component analysis of morphometric data to untangle the diversity in this taxon. Our work emphasizes the value of live animal observations that enable the identification of important characters and highlights the overlooked diversity within the Stilbonematinae