[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 of London. Series B, Biological sciences. 07/2014; 369(1647).
[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 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, 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. · 3.53 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 via 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(-1). The results of the crystal characterization are suitable for either optimal planning of room-temperature data collection or in situ 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.
Journal of inorganic biochemistry 07/2012; 115:174-81. · 3.25 Impact Factor
[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.
Journal of Molecular Biology 02/2010; · 3.91 Impact Factor
[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.
Journal of Molecular Biology 05/2009; 389(5):846-62. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.
[Show abstract][Hide abstract] ABSTRACT: The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.
Journal of Molecular Biology 01/2007; 364(3):411-23. · 3.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The import motor of the mitochondrial translocase of the inner membrane (TIM23) mediates the ATP-dependent translocation of preproteins into the mitochondrial matrix by cycles of binding to and release from mtHsp70. An essential step of this process is the stimulation of the ATPase activity of mtHsp70 performed by the J cochaperone Tim14. Tim14 forms a complex with the J-like protein Tim16. The crystal structure of this complex shows that the conserved domains of the two proteins have virtually identical folds but completely different surfaces enabling them to perform different functions. The Tim14-Tim16 dimer reveals a previously undescribed arrangement of J and J-like domains. Mutations that destroy the complex between Tim14 and Tim16 are lethal demonstrating that complex formation is an essential requirement for the viability of cells. We further demonstrate tight regulation of the cochaperone activity of Tim14 by Tim16. The first crystal structure of a J domain in complex with a regulatory protein provides new insights into the function of the mitochondrial TIM23 translocase and the Hsp70 chaperone system in general.
The EMBO Journal 11/2006; 25(19):4675-85. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel cytochrome c nitrite reductase (TvNiR) was isolated from the haloalkalophilic bacterium Thioalkalivibrio nitratireducens. The enzyme catalyses nitrite and hydroxylamine reduction, with ammonia as the only product of both reactions. It consists of 525 amino-acid residues and contains eight haems c. TvNiR crystals were grown by the hanging-drop vapour-diffusion technique. The crystals display cubic symmetry and belong to space group P2(1)3, with unit-cell parameter a = 194 A. A native data set was obtained to 1.5 A resolution. The structure was solved by the SAD technique using the data collected at the Fe absorption peak wavelength.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2006; 62(Pt 3):215-7. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Aminopeptidase T (AmpT) from Thermus thermophilus is a metalloexopeptidase with no similarity to prototypical metallopeptidases with an HExxH or HxxEH motif. The crystal structure of the Staphylococcus aureus homologue of AmpT, which is known as aminopeptidase S (AmpS), has been reported recently. This structure revealed a dimeric protein with a very unusual, elongated shape and a large internal cavity. The active sites were found on the inner walls of the cavity and were entirely shielded from the environment, which suggested either that the dimer in the crystals was not physiologically relevant, or that an inactive conformation had been crystallized. Here, we show by gel-filtration and analytical ultracentrifugation that AmpT, like AmpS, forms dimers in solution, and we present the structure of AmpT in a crystal form with five protomers in the asymmetric unit. The five protomers take conformations that range from fully closed, as in the AmpS structure, to nearly open, so that the active site is almost directly accessible. The different conformations indicate flexibility between the AmpT N and C-domains, and explain how AmpT can be active, although the unusual AmpS dimerization mode applies to AmpT as well.
Journal of Molecular Biology 11/2005; 354(2):403-412. · 3.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Triple-helical collagen IV protomers associate through their N- and C-termini, forming a three-dimensional network that provides basement membranes with mechanical strength. Within this network, the C-terminal non-collagenous (NC1) domains form tight dimeric junctions. Crystallographic analyses of isolated NC1 domains show two trimeric cap-like structures interacting via a large interface. Previously, for NC1 from human placenta type-IV collagen we described covalent alpha1-alpha1 and alpha2-alpha2 crosslinks between Met93 and Lys211 of opposing alpha1(IV) and alpha2(IV) NC1-chains, which further stabilize this interface and explain the occurrence of reduction-insensitive NC1-chain dimers. However, their existence was recently questioned, and we therefore analyzed NC1-domain dimers in more detail by biochemical and protein crystallographic methods. Short-exposure diffraction data show a clear electron density cross-connecting the respective residues, which gradually disappears with prolonged crystal irradiation. Sequence analyses of isolated tryptic peptides derived from denatured NC1 monomers and dimers indicate that only the dimers, but not the monomers, yield these chemically labile cross-linked peptides. These data clearly demonstrate the presence of reduction-resistant, but chemically and radiation-sensitive covalent crosslinks between the side chains of Met93 and Lys211 in human placenta type-IV collagen.