Alexander N. Popov

EDNA: a framework for plugin-based applications applied to X-ray experiment online data analysis.

EDNA is a framework for developing plugin-based applications especially for online data analysis in the X-ray experiments field. This article describes the features provided by the EDNA framework to ease the development of extensible scientific applications. This framework includes a plugins class hierarchy, configuration and application facilities, a mechanism to generate data classes and a testing framework. These utilities allow rapid development and integration in which robustness and quality play a fundamental role. A first prototype, designed for macromolecular crystallography experiments and tested at several synchrotrons, is presented.

Three-dimensional structure of the enzyme dimanganese catalase from Thermus Thermophilus at 1 Å resolution

The crystal structures of two forms of the enzyme dimanganese catalase from Thermus Thermophilus (native and inhibited by chloride) were studied by X-ray diffraction analysis at 1.05 and 0.98 Å resolution, respectively. The atomic models of the molecules were refined to the R factors 9.8 and 10%, respectively. The three-dimensional molecular structures are characterized in detail. The analysis of electron-density distributions in the active centers of the native and inhibited enzyme forms revealed that the most flexible side chains of the amino acid residues Lys162 and Glu36 exist in two interrelated conformations. This allowed us to obtain the structural data necessary for understanding the mechanism of enzymatic activity of the dimanganese catalase.

A quantitative approach to data-collection strategies

Statistical descriptors of the X-ray diffraction data set for a macromolecular crystal can be modelled using the information present in the initial diffraction images. Quantitative relationships between the crystal quality, beam apertures, oscillation width, resolution limit, redundancy and the data statistics are presented. They are analysed in terms of the radiation-dose requirements based on modelling in program the BEST. The influence of radiation damage on the data statistics is discussed.

Choice of data-collection parameters based on statistic modelling.

A new method and the software program BEST for optimal planning of X-ray data collection from protein crystals using the rotation method are presented. From one or a few initial diffraction images, BEST estimates the statistical characteristics of the data set for different combinations of data-collection parameters and suggests the most optimal ones. The anisotropy in diffraction and the permitted width of oscillation without spatially overlapping reflections are taken into account. According to the option chosen, the optimal set of parameters provides a given average signal-to-noise ratio at a given resolution either in the shortest time or with the minimum total radiation dose. BEST has been successfully used at the protein crystallography beamlines at DORIS (DESY). The software proved to be extremely useful in using the available data-collection time in the most efficient way.

The application of hierarchical cluster analysis to the selection of isomorphous crystals.

It is generally assumed that the quality of X-ray diffraction data can be improved by merging data sets from several crystals. However, this effect is only valid if the data sets used are from crystals that are structurally identical. It is found that frozen macromolecular crystals very often have relatively low structure identity (and are therefore not isomorphous); thus, to obtain a real gain from multi-crystal data sets one needs to make an appropriate selection of structurally similar crystals. The application of hierarchical cluster analysis, based on the matrix of the correlation coefficient between scaled intensities, is proposed for the identification of isomorphous data sets. Multi-crystal single-wavelength anomalous dispersion data sets from four different protein molecules have been probed to test the applicability of this method. The use of hierarchical cluster analysis permitted the selection of batches of data sets which when merged together significantly improved the crystallographic indicators of the merged data and allowed solution of the structure.

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.

Non-covalent interactions in the crystallization of the enantiomers of 1,7-dioxaspiro[5.5]undecane (olive fly sex pheromone) by enantiospecific cyclodextrin hosts, hexakis(2,3,6-tri-O-methyl)-α-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin

The enantiomers of racemic olive fly sex pheromone 1,7-dioxaspiro[5.5]undecane (1) have been isolated by crystallization with enantiospecific cyclodextrin hosts: (S)-(1) crystallizes with heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) and (R)-(1) with hexakis(2,3,6-tri-O-methyl)-alpha-cyclodextrin (TMalphaCD). The crystal structure of TMbetaCD/(S)-(1) from synchrotron radiation data at 100 K, determined for the first time, proves that TMbetaCD crystallizes with only the (S)-enantiomer from the racemic mixture. Comparison with the 100 K structure of TMalphaCD/(R)-(1) redetermined with synchrotron data has provided insight into the interactions between each of the hosts with the corresponding enantiomeric guests. Owing to the high resolution of the data and the unusually high quality of the crystals, localization of the H atoms has been achieved, a rare accomplishment for cyclodextrin X-ray structures. This made possible, apart from the geometry of the complexes, the detailed description of a five-membered-ring water cluster with very well ordered hydrogen bonding. The enantiospecificity exhibited by the described systems reveals the subtle differences of the weak intermolecular forces involved in the selective binding of the two optical antipodes by the two hosts. The binding geometry in the two complexes is different, but it is effected in both by numerous host-guest C-H.O interactions, resulting from induced fit of the hosts toward each of the enantiomeric guests. In TMalphaCD/(R)-(1) two of these H.O host-guest distances, directed toward the acetal O atoms defining the chirality of the guest, are much shorter than the rest and also shorter than all the H.O distances in TMbetaCD/(S)-(1). Moreover, (R)-(1) interacts not only with the enclosing host, but with other hosts in the crystal lattice, in contrast to (S)-(1) in the TMbetaCD/(S)-(1) complex which is isolated inside channels formed by the host molecules. The above differences are reflected in the much higher binding constant of TMalphaCD/(R)-(1) compared with that of TMbetaCD/(S)-(1) ( approximately 6800 and approximately 935 M(-1), respectively), determined by NMR in aqueous solution, and the ability of TMalphaCD to selectively precipitate (R)-(1) from racemic (1) in much higher yield than TMbetaCD precipitates (S)-(1).

A Bayesian approach to Laue diffraction analysis and its potential for time-resolved protein crystallography

A solution to the energy-overlap problem in Lane diffraction is described that does not require redundancy in the measurements. The new method follows a Bayesian approach with multidimensional probability density functions. The only assumption made is the validity of Wilson statistics. 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 Bayesian method may also be applied to deconvoluting spatial overlaps. The power of the method is demonstrated by a test application to bovine trypsin. The completeness at low and medium resolution as well as at very high resolution (1.4u2005A) is enhanced very substantially as compared with standard procedures; the `low-resolution hole problem is solved. As a consequence, the contrast in electron-density maps improves so far that they become comparable in quality with maps from monochromatic data at high 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. Simulation calculations for single-shot Laue conditions and for the disorder-order transition in trypsinogen as a model system demonstrate the potential power of applications in protein crystallography that combine high resolution and Bayesian processing.

Improving the X-ray resolution by reversible flash-cooling combined with concentration screening, as exemplified with PPase

A significant improvement in the X-ray resolution of crystals of Escherichia coli inorganic pyrophosphatase at cryotemperature was obtained as a result of studying the relationship between the crystal order and cryosolution component concentrations. To perform the experiments, the ability to reverse the flash-cooling process and to return a crystal to ambient temperature was used. In each cycle, the crystal was transferred from a cold nitrogen-gas stream to a cryosolution with modified concentrations of the components. The crystal was then flash-cooled again and the diffraction quality checked. Such a technique allows the screening of a wide concentration range rather quickly without using a large number of crystals and allows the determination of optimal cryosolution component concentrations. The resolution limit for crystals of pyrophosphatase increased by almost 0.7 A, from 1.8 to 1.15 A.

A survey of global radiation damage to 15 different protein crystal types at room temperature: a new decay model

A systematic study of the sensitivity to radiation damage of crystals held at room temperature for a large set of model macromolecular structures is presented.