Molecular basis for amyloid-β polymorphism Colletier, Jacques-Philippe; Laganowsky, Arthur; Landau, Meytal ...
Proceedings of the National Academy of Sciences,
10/2011, Letnik:
108, Številka:
41
Journal Article
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Amyloid-beta (Aβ) aggregates are the main constituent of senile plaques, the histological hallmark of Alzheimer’s disease. Aβ molecules form β-sheet containing structures that assemble into a variety ...of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities. This polymorphic nature of Aβ has frustrated its biophysical characterization, its structural determination, and our understanding of its pathological mechanism. To elucidate Aβ polymorphism in atomic detail, we determined eight new microcrystal structures of fiber-forming segments of Aβ. These structures, all of short, self-complementing pairs of β-sheets termed steric zippers, reveal a variety of modes of self-association of Aβ. Combining these atomic structures with previous NMR studies allows us to propose several fiber models, offering molecular models for some of the repertoire of polydisperse structures accessible to Aβ. These structures and molecular models contribute fundamental information for understanding Aβ polymorphic nature and pathogenesis.
The first phase of the ESRF beamline ID23 to be constructed was ID23‐1, a tunable MAD‐capable beamline which opened to users in early 2004. The second phase of the beamline to be constructed is ...ID23‐2, a monochromatic microfocus beamline dedicated to macromolecular crystallography experiments. Beamline ID23‐2 makes use of well characterized optical elements: a single‐bounce silicon (111) monochromator and two mirrors in Kirkpatrick–Baez geometry to focus the X‐ray beam. A major design goal of the ID23‐2 beamline is to provide a reliable, easy‐to‐use and routine microfocus beam. ID23‐2 started operation in November 2005, as the first beamline dedicated to microfocus macromolecular crystallography. The beamline has taken the standard automated ESRF macromolecular crystallography environment (both hardware and software), allowing users of ID23‐2 to be rapidly familiar with the microfocus environment. This paper describes the beamline design, the special considerations taken into account given the microfocus beam, and summarizes the results of the first years of the beamline operation.
ID23‐2 is a fixed‐energy (14.2 keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have ...recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF‐EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013 photons s−1) focused microbeam (minimum size 1.5 µm × 3 µm full width at half‐maximum). The sample environment now includes a FLEX‐HCD sample changer/storage system, as well as a state‐of‐the‐art MD3Up high‐precision multi‐axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments.
ID23‐2 is a microfocus synchrotron beamline dedicated to macromolcular crystallography at the ESRF‐EBS.
Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many ...years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF‐1 (ID30A‐1), based on an industrial six‐axis robot, is described. The device, named RoboDiff, includes a high‐capacity dewar, acts as both a sample changer and a high‐accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF‐1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.
An industrial six‐axis robot has been combined with a high‐accuracy air‐bearing rotation axis to create a single device with the capabilities of both transferring cryocooled protein crystals from a sample‐containing dewar and collecting complete X‐ray diffraction data sets.
The design and features of a beamline control software system for macromolecular crystallography (MX) experiments developed at the European Synchrotron Radiation Facility (ESRF) are described. This ...system, MxCuBE, allows users to easily and simply interact with beamline hardware components and provides automated routines for common tasks in the operation of a synchrotron beamline dedicated to experiments in MX. Additional functionality is provided through intuitive interfaces that enable the assessment of the diffraction characteristics of samples, experiment planning, automatic data collection and the on‐line collection and analysis of X‐ray emission spectra. The software can be run in a tandem client‐server mode that allows for remote control and relevant experimental parameters and results are automatically logged in a relational database, ISPyB. MxCuBE is modular, flexible and extensible and is currently deployed on eight macromolecular crystallography beamlines at the ESRF. Additionally, the software is installed at MAX‐lab beamline I911‐3 and at BESSY beamline BL14.1.
A revised version of Table 2 of Nanao et al. J. Synchrotron Rad. (2022). 29, 581–590 is provided.
Corrections to Table 2 of Nanao et al. J. Synchrotron Rad. (2022). 29, 581–590 are reported.
Crystals of biological macromolecules often exhibit considerable inter‐crystal and intra‐crystal variation in diffraction quality. This requires the evaluation of many samples prior to data ...collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron‐based facilities optimized for high‐throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample‐containing loop for microcrystals using an X‐ray beam of 5 µm diameter and selecting the most ordered regions of relatively large crystals using X‐ray beams of 5–50 µm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction‐quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.
Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize ...membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation.
Anionic calix4arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein.
These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.
The structure of a synthetic potassium birnessite (KBi) obtained as a finely dispersed powder by thermal decomposition of KMnO4 at 800 °C was for the first time studied by single-crystal X-ray ...diffraction (XRD). It is shown that KBi has a two-layer cell with a = 2.840(1) Å and c = 14.03(1) Å and space group P63/mmc. In contrast to the structure model proposed by Kim et al. (Chem. Mater. 1999, 11, 557−563), the refined model demonstrates the sole presence of Mn4+ in the octahedral layers, the presence of 0.12 vacant layer sites per octahedron being responsible for the layer charge deficit. In agreement with X-ray absorption spectroscopy result, this layer charge deficit is compensated (1) by the presence of interlayer Mn3+ above or below vacant layer octahedra sharing three Olayer atoms with neighboring Mnlayer octahedra to form a triple-corner surface complex (VITC sites) and (2) by the presence of interlayer K in prismatic cavities located above or below empty tridentate cavities, sharing three edges with neighboring Mnlayer octahedra (VITE sites). As compared to the structure model proposed by Kim et al., this VITE site is shifted from the center of the prismatic cavity toward its edges. A complementary powder XRD study confirmed the structure model of the main defect-free KBi phase and allowed for the determination of the nature of the stacking disorder in a defective accessory KBi phase admixed to the defect-free KBi.
The pathogenic bacterium Shigella flexneri uses a type III secretion system to inject virulence factors from the bacterial cytosol directly into host cells. The machinery that identifies secretion ...substrates and controls the export of extracellular components and effector proteins consists of several inner-membrane and cytoplasmic proteins. One of the inner membrane components, Spa40, belongs to a family of proteins proposed to regulate the switching of substrate specificity of the export apparatus. We show that Spa40 is cleaved within the strictly conserved amino acid sequence NPTH and substitution of the proposed autocatalytic residue abolishes cleavage. Here we also report the crystal structure of the cytoplasmic complex Spa40C and compare it with the recent structures of the homologues from Escherichia coli and Salmonella typhimurium. These structures reveal the tight association of the cleaved fragments and show that the conserved NPTH sequence lies on a loop which, when cleaved, swings away from the catalytic N257 residue, resulting in different surface features in this region. This structural rearrangement suggests a mechanism by which non-cleaving forms of these proteins interfere with correct substrate switching of the apparatus.
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