[Show abstract][Hide abstract] ABSTRACT: Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.
[Show abstract][Hide abstract] ABSTRACT: Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.1 Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.2-6 AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg(2+) as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg(2+) and in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.
[Show abstract][Hide abstract] ABSTRACT: Protein crystallography continues to be one of the most frequently used techniques to obtain structural information of biomacromolecules to atomic resolution. Since protein crystals of delicate target systems are often limited in size, one of the main goals in the design of modern beamlines is the construction of highly intense X-ray beams with small focal size to obtain high resolution diffraction images of microcrystals. However, this development has led to the situation, that the full intensity of the beam can destroy a protein crystal within fractions of a second. Therefore often only a small number of diffraction patterns can be obtained from one single crystal. Here we describe the adaptation of the serial crystallography approach, which has first been developed at X-ray Free-Electron Lasers (Chapman et al. 2011) to the usage of a microfocus synchrotron beamline, using a standard cryogenic loop for sample delivery. We proved this concept with in vivo grown cathepsinB microcrystals (TbCatB, Koopmann et al. 2012, Redecke et al. 2013) (average of 9 μm3), a medically and pharmaceutically relevant protein, involved in the life cycle of T. brucei. In these experiments it was possible to show that serial crystallography enables the utilization and outcome of the above described bottlenecks and features of modern 3rd generation synchrotron microfocus beamlines. Our strategy exploits the combination of a micron-sized X-ray beam, high precision diffractometry and shutterless data acquisition with a pixel-array detector. By combining the data of 80 TbCatB crystals, it was possible to assemble a dataset to 3.0 Å resolution. The data allow the refinement of a structural model that is consistent with that previously obtained using FEL radiation, providing mutual validation.
Acta Crystallographica Section A: Foundations and Advances 08/2014; 70(a1):C316-C316. DOI:10.1107/S2053273314096831
[Show abstract][Hide abstract] ABSTRACT: Serial crystallography using X-ray free-electron lasers enables the collection of tens of thousands of measurements from an equal number of individual crystals, each of which can be smaller than 1 µm in size. This manuscript describes an alternative way of handling diffraction data recorded by serial femtosecond crystallography, by mapping the diffracted intensities into three-dimensional reciprocal space rather than integrating each image in two dimensions as in the classical approach. We call this procedure 'three-dimensional merging'. This procedure retains information about asymmetry in Bragg peaks and diffracted intensities between Bragg spots. This intensity distribution can be used to extract reflection intensities for structure determination and opens up novel avenues for post-refinement, while observed intensity between Bragg peaks and peak asymmetry are of potential use in novel direct phasing strategies.
Philosophical Transactions of The Royal Society B Biological Sciences 07/2014; 369(1647). DOI:10.1098/rstb.2013.0333 · 7.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80
procathepsin B crystals with an average volume of 9 µm
, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.
[Show abstract][Hide abstract] ABSTRACT: Copper-containing ferroxidase ceruloplasmin (Cp) forms binary and ternary complexes with cationic proteins lactoferrin (Lf) and myeloperoxidase (Mpo) during inflammation. We present an X-ray crystal structure of a 2Cp-Mpo complex at 4.7 Å resolution. This structure allows one to identify major protein-protein interaction areas and provides an explanation for a competitive inhibition of Mpo by Cp and for the activation of p-phenylenediamine oxidation by Mpo. Small angle X-ray scattering was employed to construct low-resolution models of the Cp-Lf complex and, for the first time, of the ternary 2Cp-2Lf-Mpo complex in solution. The SAXS-based model of Cp-Lf supports the predicted 1∶1 stoichiometry of the complex and demonstrates that both lobes of Lf contact domains 1 and 6 of Cp. The 2Cp-2Lf-Mpo SAXS model reveals the absence of interaction between Mpo and Lf in the ternary complex, so Cp can serve as a mediator of protein interactions in complex architecture. Mpo protects antioxidant properties of Cp by isolating its sensitive loop from proteases. The latter is important for incorporation of Fe(3+) into Lf, which activates ferroxidase activity of Cp and precludes oxidation of Cp substrates. Our models provide the structural basis for possible regulatory role of these complexes in preventing iron-induced oxidative damage.
PLoS ONE 07/2013; 8(7):e67145. DOI:10.1371/journal.pone.0067145 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The radiation damage rates to crystals of 15 model macromolecular structures were studied using an automated radiation sensitivity characterization procedure. The diffracted intensity variation with dose is described by a two-parameter model. This model includes a strong resolution-independent decay specific to room-temperature measurements along with a linear increase in overall Debye–Waller factors. An equivalent representation of sensitivity
a single parameter, normalized half-dose, is introduced. This parameter varies by an order of magnitude between the different structures studied. The data show a correlation of crystal radiation sensitivity with crystal solvent content but no dose-rate dependency was detected in the range 0.05–300 kGy s
. The results of the crystal characterization are suitable for either optimal planning of room-temperature data collection or
crystallization plate screening experiments.
[Show abstract][Hide abstract] ABSTRACT: Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A reliable and reproducible method to automatically characterize the radiation sensitivity of macromolecular crystals at the ESRF beamlines has been developed. This new approach uses the slope of the linear dependence of the overall isotropic B-factor with absorbed dose as the damage metric. The method has been implemented through an automated procedure using the EDNA on-line data analysis framework and the MxCuBE data collection control interface. The outcome of the procedure can be directly used to design an optimal data collection strategy. The results of tests carried out on a number of model and real-life crystal systems are presented.
[Show abstract][Hide abstract] ABSTRACT: To take into account the effects of radiation damage, new algorithms for the optimization of data-collection strategies have been implemented in the software package BEST. The intensity variation related to radiation damage is approximated by log-linear functions of resolution and cumulative X-ray dose. Based on an accurate prediction of the basic characteristics of data yet to be collected, BEST establishes objective relationships between the accessible data completeness, resolution and signal-to-noise statistics that can be achieved in an experiment and designs an optimal plan for data collection.
[Show abstract][Hide abstract] ABSTRACT: The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-l-nicotine oxidase (6HLNO) and 6-hydroxy-d-nicotine oxidase, which act with absolute stereospecificity on the l- and d-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 Å resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit–subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-l-nicotine at 2.05 Å resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-d-nicotine oxidase, based on models of complexes with the d-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.