22 research outputs found

    UV LED lighting for automated crystal centring

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    A low-cost light-emitting diode (LED) UV source has been developed for facilitating macromolecular sample centring in the X-ray beam

    High-pressure-induced water penetration into 3-­isopropylmalate dehydrogenase

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    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

    Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

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    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.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikEC/H2020/664726/EU/EMBL Interdisciplinary, International and Intersectorial Postdocs/EI3PO

    Structural and Biochemical Studies on Glycoconjugate Alteration and Vesicle Transport

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    Structure Determination of Human Cytoplasmic Neu2  To date, l3 mammalian sialidases have been cloned including four from humans. So far, only some bac-terial and viral neuraminidase\u27s structures are known, nothing from human or mammals. In this study, the crystal structure of the human cytosolic sialidase Neu2 was determined at the atomic level, either in an apo form or in complex with diverse inhibitors of substrate of reaction.  The core of the enzyme folds as a six-bladed β-propeller with 26 β-strands and five α-helices, typical of viral and bacterial sialidases. Neu2 adopts an irregular six-bladed structure caused by β-bulges of the third strand, as well as longer β-stands compared to the "classical" propeller foldings.  In the Neu2-DANA complex structure, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lies in a half-chair conformation interacting with 10 amino acids of the active site. Interaction between Neu2 and the inhibitor DANA shows similarities with bacterial and viral counterparts but also exhibits some differences in the active site arrangement and dynamic nature of the loops containing residues respon-sible for catalysis and substrate recognition An acidic crevice at the center of the β-propeller forms an activating core for the enzymatic catalysis and the basic residues at the mouth of the crevice coordinate substrates.  Two loops are disordered on the top side of the apo form Neu2: one contains Glu111 important for the substrate binding, and the other Asp46 for the catalysis. Soaking the apo form crystals with monosaccharide such as galactose, glucose, or maltose orders the former loop into α2, illustrating a plausible two steps model for a dynamic in the substrate recognition. In the Neu2 structures, two Asp-boxes STDHGRTW (residues 129-136) and SHDHGRTW (residues 199-206) are easily identified (where boldface letters indicate residues structurally important for Asp-boxes). A third Asp-box STNDGLDF (residues 248-255) was found to have similar folding property comparing with the two firsts. The sequence of the third Asp-box in Neu2 extends the consensus se-quence of Asp-boxes as Ser-X-(Asp/Gln)-X-Gly-X-(Thr/Asp)-〓, where 〓stands for aromatic residues. A new screening may therefore give further insights into the function of these structural elements.  Finally, different crystal forms of the sialidase have been refined in order to facilitate inhibitors screenings. The influenza virus neuraminidase plays a critical role in the life cycle of the virus and has been the focus of new drug developments. Drug design of new agents against the viral sialidase comes from the fact that whereas diverse kinds of influenza virus neuraminidases were identified, their catalytic site is completely conserved among all influenza subtypes in terms of amino acids organization. To date, no data exist on whether the human sialidases can interact as well with viral and/or bacterial targeting drugs. To answer this question, complex structures of Neu2 with overall six different influenza virus in-hibitors have been solved, illustrating the inhibitors lying in the human sialidase active site coordinated in a similar way. By comparing tle recognition mechanism of each inhibitor, in Neu2 or in influenza virus neuraminidase, new drug design studies might be started in order to preferentially recognize either human or viral sialidases.  Biochemical and Crystallographic Studies on Rab27a/b and Their Effectors  The integrity of eukaryotic cells stongly depends on membrane traffic, which is tightly linked to the regulation by a number of protein families such as Ras, Rho, Rab and Ran. Those proteins bind to var-ious types of effector proteins, and execute versatile functions. More precisely, the Rab27 subfamily, consisting of Rab27a and Rab27b, is directly implicated in the hansport of lysosome-related organelles, such as melanosomes in melanocytes or lytic granules in cytotoxic T-lymphorytes. In order to clarify the regulation process of such transport phenomena, biochemical and crystallization experiments have been carried out on both Rab27a/b, alone and in complex with two effectors Slac2-a and Slp4-a.  The individual GTPases, Rab27a and Rab27b, were purified for suitable crystallization experiments, both in their active (GTP-bound) or inactive (GDP-bound)forms. Extensive crystallization screenings have been performed, resulting in the apparition of some protein diffracting crystals in diverse conditions for Rab27b in its inactive form. The crystals grew in a tetragonal space group but did not diffract at higher resolution than 4.2 Å. Seleno-methionine substituted Rab27b protein was then purified and crystallized, but the crystal\u27s diffraction limit did not improve. Further refinements in terms of protein deletion mutants and crystallization are now under way in order to obtain highly diffracting crystals.  The complexes between the GTPases and their effectors, respectively Slac2-a and Slp4-a, could be generated and purified, and have been subjects of intensive crystallization trials with the help of a crystallization robot. Unfortunately, to date no protein crystals suitable for X-ray structure determination have been obtained

    Manufacturing of Ultra-Thin X-ray-Compatible COC Microfluidic Devices for Optimal In Situ Macromolecular Crystallography Experiments

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    Cyclic-olefin-copolymer (COC)-based microfluidic devices are increasingly becoming the center of highly valuable research for in situ X-ray measurements due to their compatibility with X-rays, biological compounds, chemical resistance, optical properties, low cost, and simplified handling. COC microfluidic devices present potential solutions to challenging biological applications such as protein binding, folding, nucleation, growth kinetics, and structural changes. In recent years, the techniques applied to manufacturing and handling these devices have capitalized on enormous progress toward small-scale sample probing. Here, we describe the new and innovative design aspects, fabrication, and experimental implementation of low-cost and micron-sized X-ray-compatible microfluidic sample environments that address diffusion-based crystal formation for crystallographic characterization. The devices appear fully compatible with crystal growth and subsequent X-ray diffraction experiments, resulting in remarkably low background data recording. The results highlighted in this research demonstrate how the engineered microfluidic devices allow the recording of accurate crystallographic data at room temperature and structure determination at high resolution
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