Search

A microfluidic laboratory recently opened at Synchrotron SOLEIL, dedicated to in-house research and external users. Its purpose is to provide the equipment and expertise that allow the development of microfluidic systems adapted to the beamlines of SOLEIL as well as other light sources. Such systems can be used to continuously deliver a liquid sample under a photon beam, keep a solid sample in a liquid environment or provide a means to track a chemical reaction in a time-resolved manner. The laboratory provides all the amenities required for the design and preparation of soft-lithography microfluidic chips compatible with synchrotron-based experiments. Three examples of microfluidic systems that were used on SOLEIL beamlines are presented, which allow the use of X-ray techniques to study physical, chemical or biological phenomena.

By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3–6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-l-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.

Description, A bacterial phytochrome reversibly interconverts between a parallel and antiparallel dimeric arrangement during photoconversion., Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light–absorbing) and Pfr (far-red light–absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.

An approach for serial crystallography experiments based on wedged-data collection is described. This is an alternative method for recording in situ X-ray diffraction data on crystalline samples efficiently loaded in an X-ray compatible microfluidic chip. Proper handling of the microfluidic chip places crystalline samples at geometrically known positions with respect to the focused X-ray interaction area for serial data collection of small wedges. The integration of this strategy takes advantage of the greatly modular sample environment available on the endstation, which allows access to both in situ and more classical cryo-crystallography with minimum time loss. The method represents another optional data collection approach that adds up to the already large set of methods made available to users. Coupled with the advances in processing serial crystallography data, the wedged-data collection strategy proves highly efficient in minimizing the amount of required sample crystals for recording a complete dataset. From the advances in microfluidic technology presented here, high-throughput room-temperature crystallography experiments may become routine and should be easily extended to industrial use.

By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas

Red/far-red light-sensing bacteriophytochrome photoreceptor (BphP) pathways play key roles in bacterial physiology and ecology. These bilin-binding proteins photoswitch between two states, Pr (red absorbing) and Pfr (far-red absorbing). The isomerization of the chromophore and the downstream structural changes result in the light signal transduction. The agricultural pathogen Xanthomonas campestris pv. campestris (Xcc) code for a single bathy-like type BphP (XccBphP), previously shown to negatively regulate several light-mediated biological processes involved in virulence. Here, we generated three different full-length variants with single amino acid changes within its GAF domain that affect the XccBphP photocycle favouring its Pr state: L193Q, L193N and D199A. While D199A recombinant protein locks XccBphP in a Pr-like state, L193Q and L193N exhibit a significant enrichment of the Pr form in thermal equilibrium. The X-ray crystal structures of the three variants were solved, resembling the wild-type protein in the Pr state. Finally, we studied the effects of altering the XccBphP photocycle on the exopolysaccharide (EPS) xanthan production and stomatal aperture assays as readouts of its bacterial signaling pathway. Null-mutant complementation assays show that the photoactive Pr-favoured XccBphP variants L193Q and L193N tend to negatively regulate xanthan production in vivo. In addition, our results indicate that strains expressing these variants also promote stomatal apertures in challenged plant epidermal peels, compared to wild-type Xcc. The findings presented in this work provide new evidence on the Pr state of XccBphP as a negative regulator of the virulence-associated mechanisms by light in Xcc.

Light sensing allows organisms to adapt to constantly changing environmental factors. Phytochromes constitute a widespread biological photoreceptor family that typically interconvert between two photostates called Pr (red light-absorbing) and Pfr (far-red light-absorbing). Despite the vast structural information reported on phytochromes, the lack of full-length structures at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here we present three crystallographic structures from the plant pathogenXanthomonas campestrisvirulence regulator bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structural findings, combined with mutational, biochemical and computational studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level, from the isomerization of the chromophore and the β-sheet/α-helix tongue transition to the remodeling of the quaternary assembly of the protein.TEASERCrystal structures of the full-length bacteriophytochromeXccBphP in both Pr and Pfr states unveil photoswitching mechanism

PROXIMA-1 at Synchrotron SOLEIL has been run for 12 years as an endstation dedicated to macromolecular crystallography experiments, optimized for anomalous data recording and diffraction studies of large unit-cell crystals., The undulator beamline PROXIMA-1 at Synchrotron SOLEIL scheduled its first users in March 2008. The endstation is dedicated to biomolecular crystallography experiments, with a layout designed to favour anomalous data recording and studies of crystals with large cell dimensions. In 12 years, the beamline has accommodated 4267 shifts of 8 h and more than 6300 visitors. By the end of 2020, it saw 1039 identified published scientific papers referring to 1415 coordinates deposited in the Protein Data Bank. The current paper describes the PROXIMA-1 beamline, including the recent specific implementations developed for the sample environment. The setup installed in the experimental station contains numerous beam-shaping equipment, a chi-geometry three-axis goniometer, a single-photon-counting pixel-array X-ray detector, combined with a medium-throughput sample exchange robot. As part of a standard experimental scheme, PROXIMA-1 can also be accessed via ‘mail-in’ services or remotely.

The Pacific Beetle Cockroach is the only known viviparous cockroach. The pregnant females provide nutrition to the embryos by secreting milk proteins (Lili-Mips), which crystallize in vivo. The crystals that grow in the embryo are heterogeneous in their protein sequence. It is not apparent from the structure determined what role heterogeneity and glycosylation played in crystallization. Lili-Mips are very nutritious.Here, we report the cloning of synthesized Lili-Mip genes, their expression in Saccharomyces cerevisiae as secreted proteins, purification, crystallization, and the determination of a three-dimensional structure of one glycosylated and one deglycosylated form.A 2.35 Å structure of the glycosylated form is bound to palmitoleic acid and has several Zn atom mediated interactions. A 1.45 Å structure of the deglycosylated protein reveals a binding pocket that has both oleic and palmitoleic acid bound. Mass-spectrometry shows that oleic acid and palmitoleic acid are bound to the protein. Docking studies suggest that aliphatic chains of lengths 15, 16, and 18 carbons bind well in the pocket.The recombinantly expressed and secreted protein is glycosylated, has a bound fatty acid, is homogenous in its protein sequences, and readily forms crystals. The deglycosylated protein also crystallizes readily, suggesting that the high crystallizability of this protein is independent of glycosylation.Lili-Mips belong to the ubiquitous lipocalin family of proteins that bind to a large variety of ligands. While the residues lining the barrel are essential for the affinity of the ligand, our results show the role of side-chain orientations to ligand selectivity.

Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid’s C-4 substituent’s size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

Journal of synchrotron radiation 26(2), 393 - 405 (2019). doi:10.1107/S1600577519001267, MXCuBE2 is the second-generation evolution of the MXCuBE beamline control software, initially developed and used at ESRF – the European Synchrotron. MXCuBE2 extends, in an intuitive graphical user interface (GUI), the functionalities and data collection methods available to users while keeping all previously available features and allowing for the straightforward incorporation of ongoing and future developments. MXCuBE2 introduces an extended abstraction layer that allows easy interfacing of any kind of macromolecular crystallography (MX) hardware component, whether this is a diffractometer, sample changer, detector or optical element. MXCuBE2 also works in strong synergy with the ISPyB Laboratory Information Management System, accessing the list of samples available for a particular experimental session and associating, either from instructions contained in ISPyB or from user input via the MXCuBE2 GUI, different data collection types to them. The development of MXCuBE2 forms the core of a fruitful collaboration which brings together several European synchrotrons and a software development factory and, as such, defines a new paradigm for the development of beamline control platforms for the European MX user community., Published by Wiley-Blackwell, [S.l.]

This article presents the features and structures of protein crystals naturally grown in vivo within developing embryos of the only known viviparous cockroach, D. punctata. This study reveals the heterogeneous nature of the crystalline protein with respect to amino-acid sequence, glycosylation and bound fatty acid at atomic resolution., Macromolecular crystals for X-ray diffraction studies are typically grown in vitro from pure and homogeneous samples; however, there are examples of protein crystals that have been identified in vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown in cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These in vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, Diploptera punctata. The milk proteins crystallized in space group P1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single in vivo-grown crystal of a natural protein in its native environment at atomic resolution.

Single molecule dynamics studies have begun to use quantum probes. Single particle analysis using cryo-transmission electron microscopy has dramatically improved the resolution when studying protein structures and is shifting towards molecular motion observations. X-ray free-electron lasers are also being explored as routes for determining single molecule structures of biological entities. Here, we propose a new X-ray single molecule technology that allows observation of molecular internal motion over long time scales, ranging from milliseconds up to 103 seconds. Our method uses both low-dose monochromatic X-rays and nanocrystal labelling technology. During monochromatic X-ray diffraction experiments, the intensity of X-ray diffraction from moving single nanocrystals appears to blink because of Brownian motion in aqueous solutions. X-ray diffraction spots from moving nanocrystals were observed to cycle in and out of the Bragg condition. Consequently, the internal motions of a protein molecule labelled with nanocrystals could be extracted from the time trajectory using this diffracted X-ray blinking (DXB) approach. Finally, we succeeded in distinguishing the degree of fluctuation motions of an individual acetylcholine-binding protein (AChBP) interacting with acetylcholine (ACh) using a laboratory X-ray source.

Cytoskeleton 74(12), 472 - 481 (2017). doi:10.1002/cm.21378, A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked β-strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual α-synuclein amyloids., Published by Wiley, Bognor Regis

Journal of synchrotron radiation 24(6), 1296 - 1298 (2017). doi:10.1107/S1600577517011961, The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce backgroundscattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude., Published by IUCr, Chester

International audience; Indolone-N-oxides have antiplasmodial properties against Plasmodium falciparum at the erythrocytic stage, with IC50 values in the nanomolar range. The mechanism of action of indolone derivatives involves the production of free radicals, which follows their bioreduction by an unknown mechanism. In this study, we hypothesized that human quinone reductase 2 (hQR2), known to act as a flavin redox switch upon binding to the broadly used antimalarial chloroquine, could be involved in the activity of the redox-active indolone derivatives. Therefore, we investigated the role of hQR2 in the reduction of indolone derivatives. We analyzed the interaction between hQR2 and several indolone-type derivatives by examining enzymatic kinetics, the substrate/protein complex structure with X-ray diffraction analysis, and the production of free radicals with electron paramagnetic resonance. The reduction of each compound in cells overexpressing hQR2 was compared to its reduction in naïve cells. This process could be inhibited by the specific hQR2 inhibitor, S29434. These results confirmed that the anti-malarial activity of indolone-type derivatives was linked to their ability to serve as hQR2 substrates and not as hQR2 inhibitors as reported for chloroquine, leading to the possibility that substrate of hQR2 could be considered as a new avenue for the design of new antimalarial compounds.

A microfluidic platform was used to address the problems of obtaining diffraction-quality crystals and crystal handling during transfer to the X-ray diffractometer. Crystallization conditions of a protein of pharmaceutical interest were optimized and X-ray data were collected both in situ and ex situ.

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

International audience; Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

The BL-17A macromolecular crystallography beamline at the Photon Factory was updated to improve the accuracy of diffraction experiments conducted using tiny crystals., BL-17A is a macromolecular crystallography beamline dedicated to diffraction experiments conducted using micro-crystals and structure determination studies using a lower energy X-ray beam. In these experiments, highly accurate diffraction intensity measurements are definitively important. Since this beamline was constructed, the beamline apparatus has been improved in several ways to enable the collection of accurate diffraction data. The stability of the beam intensities at the sample position was recently improved by modifying the monochromator. The diffractometer has also been improved. A new detector table was installed to prevent distortions in the diffractometer’s base during the repositioning of the diffractometer detector. A new pinhole system and an on-axis viewing system were installed to improve the X-ray beam profile at the sample position and the centering of tiny crystal samples.

A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed., Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.

Structures of 3-­isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration., Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.