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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. [ABSTRACT FROM AUTHOR]

High-quality high-multiplicity X-ray diffraction data were collected on five different crystals of thaumatin using a homogeneous-profile X-ray beam at E = 8 keV to investigate the counteracting effects of increased multiplicity and increased radiation damage on the quality of anomalous diffraction data collected on macromolecular crystals. By comparing sulfur substructures obtained from subsets of the data selected as a function of absorbed X-ray dose with sulfur positions in the respective refined reference structures, the doses at which the highest quality of anomalous differences could be obtained were identified for the five crystals. A statistic σ{Δ F} D, calculated as the width σ of the normalized distribution of a set {Δ F} of anomalous differences collected at a dose D, is suggested as a measure of anomalous data quality as a function of dose. An empirical rule is proposed to identify the dose at which the gains in data quality due to increased multiplicity are outbalanced by the losses due to decreases in signal-to-noise as a consequence of radiation damage. Identifying this point of diminishing returns allows the optimization of the choice of data collection parameters and the selection of data to be used in subsequent crystal structure determination steps. [ABSTRACT FROM AUTHOR]

Using the signal of naturally inbuilt or artificially introduced anomalous scatterers to derive initial phases in a macromolecular crystal structure determination has become routine in recent years. In the context of high-throughput crystallography in particular, MAD and SAD (multiple- and single-wavelength anomalous dispersion) methods are central tools. For both techniques, a crucial step is the determination of the substructure of anomalous scatterers; subsequent phasing procedures will profit from a substructure model that is as accurate as possible. The choice of the subset of the diffraction data to be used for the substructure determination has a strong influence on the quality of the substructure and can make the difference between success and failure. The accuracy of selenium substructures obtained using F[subA] values or various anomalous differences truncated to different resolutions has been investigated by comparing the sites determined by SHELXD with the selenium positions in the refined models. Based on the analysis, some recommendations for obtaining accurate and precise substructures are derived. [ABSTRACT FROM AUTHOR]

The program SITCOM, which can be used to facilitate the comparison of macromolecular substructures from different sources for validation purposes and to construct a consensus model containing sites from different substructures, is described. [ABSTRACT FROM AUTHOR]