Studien zur kristallographischen Phasierung von Proteinen: Substruktur-Validierung und MAD-phasierte Elektronendichtekarten bei atomarer Auflösung

Die Röntgen-Kristallographie ist eine der wichtigsten Methoden der Strukturbiologie.Die Verfügbarkeit Schweratom-derivatisierter Proteine ermöglicht die Anwendung des kristallographischen MAD-Strukturlösungsverfahrens. Das Vorhandensein atomar aufgelöster Strukturdaten erlaubt hierbei detaillierte Aussagen über Reaktionsmechanismen von Enzymen. Aldose Reductase gehört zu einer Klasse von Enzymen, die Glucose zu Sorbitol reduzieren. Die Inhibierung des Enzyms ist relevant fuer die Behandlung von Diabetes-Folgesymptomen.In der vorliegenden Dissertation wurde die Struktur von Aldose Reductase mittels der MAD-Methode bestimmt und eine Strukturfeinerung bei 0.9 Angström Auflösung wurde mittels der experimentell (MAD-) phasierten Elektronendichtekarte bestätigt. Dabei konnten vor allem strukturelle Fehlordnungen und Wasser-Netzwerke detailliert analysiert werden.Weiterhin behandelt die vorliegende Dissertation die Entwicklung und Anwendung des Computerprogramms SitCom, welches Schweratom-Substrukturen von Proteinen, die wichtige Zwischenergebnisse der MAD- (und anderer) Methoden darstellen, systematisch vergleicht und somit einen nicht unerheblichen Beitrag zur Verbesserung der makromolekularen Strukturlösung darstellt. Die Anwendung des Programms lieferte nützliche Aussagen über empfohlene Strategien beim Gebrauch der MAD-Methode.X-ray crystallography is one of the most important methods in structural biology. The availability of heavy atom derivatives of proteins facilitates the application of the crystallographic MAD structure solution technique. Structural data of atomic resolution allow detailed statements about reaction mechanisms of enzymes. Aldose reductase belongs to a class of enzymes reducing Glucose to Sorbitol. The inhibition of this enzyme is relevant for the treatment of symptoms resulting from diabetes mellitus.In the present thesis, the structure of Aldose Reductase was determined by means of the MAD method. A structure refinement result at 0.9 Angstrom was confirmed with the experimental (MAD) electron density map. Thereby, multiple amino acid conformations and water networks were analyzed in detail.Furthermore, the thesis is concerned with the development and application of the software SitCom, which compares different heavy atom substructures of proteins systematically. The substructures are very important intermediate results of the MAD (and other) methods. Thus, SitCom contributes to the improvement of macromolecular structure solution. The application of the program yielded useful statements about recommended strategies when using the MAD technique

Online dynamic flat-field correction for MHz Microscopy data at European XFEL

The X-ray microscopy technique at the European X-ray free-electron laser (EuXFEL), operating at a MHz repetition rate, provides superior contrast and spatial-temporal resolution compared to typical microscopy techniques at other X-ray sources. In both online visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of XFEL sources hinders the use of existing flat-flied normalization methods during MHz X-ray microscopy experiments. Here, we present an online dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images. The method is used for the normalization of individual X-ray projections and has been implemented as an online analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.Comment: 14 pages, 7 figure

Evaluation of MRSAD phasing protocols

Molecular Replacement in combination with Single Anomalous Diffraction (MRSAD) is a crystallographic phasing method that can lead to structure solution starting from weak anomalous signal and/or poor MR search models, where both the SAD and MR methods alone would fail. The advent of MRSAD has been triggered by the need to reduce the MR intrinsic model bias, in particular at low resolution, and by the increasing availability of high-resolution structures for components of larger biological complexes. To explore the capabilities and limitations of currently available MRSAD protocols, we have tested these on known crystal structures where we assume that only a part of the crystallographic unit cell is known and can be placed by molecular replacement. Our model system is Cdc23NTerm, a dimeric protein of which the structure has been recently determined via S-SAD at 3.1Å resolution1 . Also, a monomeric, 1.9Å-resolution structure has been obtained in 2013 by Se-SAD2 . MRSAD has been tested on the low-resolution structure using different search models. The pipeline involves MR- and MRSAD-phasing, followed by model building/density modification with three different programs. To assess the results, the deposited PDB model has been used as a reference. The success of the protocol has been judged on the number of residues built into the electron density map, on the phase quality as evaluated via the Mean Phase Error (MPE) and real-space map correlation coefficients against the reference. Numerically, the improvements from MR to MRSAD are limited to a few degrees in terms of MPE. However, the visual inspection of electron density maps and the analysis of the real space correlation coefficients have shown that the electron densities produced with MRSAD phases are clearly superior to those produced from MR or SAD phases alone. As a next step of the study, we will apply the same strategy to larger macromolecular complexes

Online dynamic flat-field correction for MHz microscopy data at European XFEL

The high pulse intensity and repetition rate of the European X-ray Free-Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL

Ansatz für ein partnerschaftliches Forschungsdatenmanagement: Konzept für eine schleswig-holsteinische Landesinitiative zum Forschungsdatenmanagement

Das Papier „Ansatz für ein partnerschaftliches Forschungsdatenmanagement – Konzept für eine schleswig-holsteinische Landesinitiative zum Forschungsdatenmanagement“ stellt Aufgaben, Arbeitsweise, Organisation sowie Zuständigkeiten für die schleswig-holsteinische Landesinitiative zum Forschungsdatenmanagement FDM-SH vor

Experimental capabilities for liquid jet samples at sub-MHz rates at the FXE Instrument at European XFEL

The Femtosecond X-ray Experiments (FXE) instrument at the European X-ray Free-Electron Laser (EuXFEL) provides an optimized platform for investigations of ultrafast physical, chemical and biological processes. It operates in the energy range 4.7–20 keV accommodating flexible and versatile environments for a wide range of samples using diverse ultrafast X-ray spectroscopic, scattering and diffraction techniques. FXE is particularly suitable for experiments taking advantage of the sub-MHz repetition rates provided by the EuXFEL. In this paper a dedicated setup for studies on ultrafast biological and chemical dynamics in solution phase at sub-MHz rates at FXE is presented. Particular emphasis on the different liquid jet sample delivery options and their performance is given. Our portfolio of high-speed jets compatible with sub-MHz experiments includes cylindrical jets, gas dynamic virtual nozzles and flat jets. The capability to perform multi-color X-ray emission spectroscopy (XES) experiments is illustrated by a set of measurements using the dispersive X-ray spectrometer in von Hamos geometry. Static XES data collected using a multi-crystal scanning Johann-type spectrometer are also presented. A few examples of experimental results on ultrafast time-resolved X-ray emission spectroscopy and wide-angle X-ray scattering at sub-MHz pulse repetition rates are given