1,721,045 research outputs found
P13 and P14, the EMBL Beamlines for Macromolecular Crystallography at PETRA III
No full textEMBL is operating two beamlines for macromolecular
crystallography on PETRA III (DESY, Hamburg). Both
beamlines are fully tunable and provide a wide range of
beam conditions. High flux X-ray beams with adjustable
dimensions between 5 and 200 μm are available in the
energy range between 4 and 18 keV. To demonstrate the
capability of the beamlines, we will describe and discuss
typical experiments including:
- Structure solution via S-SAD phasing using 4 keV
X-rays on P13.
- Structure solution using SAD phasing at 6.5 keV on
multiple crystals with linear dimensions < 10 μm.
- Structure solution by molecular replacement from
data collected using serial helical scans on micro-crystals
presented to the beam at room temperature in
CrystalDirectTM plates.
- Rapid ( < 3 min) data collection using a
CRL-collimated X-ray beam with a ‘top-hat’ profile.
As a CrystalDirect Harvester system will be installed at
EMBL Hamburg in June 2016, we hope to be able to
present first results with crystals harvested with this
system by the time of the meeting
The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design
Full text linkThe proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics
P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high-and low-energy phasing with variable beam focusing
Full text linkThe macromolecular crystallography P13 beamline is part of the European Molecular Biology Laboratory Integrated Facility for Structural Biology at PETRA III (DESY, Hamburg, Germany) and has been in user operation since mid-2013. P13 is tunable across the energy range from 4 to 17.5 keV to support crystallographic data acquisition exploiting a wide range of elemental absorption edges for experimental phase determination. An adaptive Kirkpatrick–Baez focusing system provides an X-ray beam with a high photon flux and tunable focus size to adapt to diverse experimental situations. Data collections at energies as low as 4 keV (λ = 3.1 Å) are possible due to a beamline design minimizing background and maximizing photon flux particularly at low energy (up to 1011 photons s−1 at 4 keV), a custom calibration of the PILATUS 6M-F detector for use at low energies, and the availability of a helium path. At high energies, the high photon flux (5.4 × 1011 photons s−1 at 17.5 keV) combined with a large area detector mounted on a 2θ arm allows data collection to sub-atomic resolution (0.55 Å). A peak flux of about 8.0 × 1012 photons s−1 is reached at 11 keV. Automated sample mounting is available by means of the robotic sample changer `MARVIN' with a dewar capacity of 160 samples. In close proximity to the beamline, laboratories have been set up for sample preparation and characterization; a laboratory specifically equipped for on-site heavy atom derivatization with a library of more than 150 compounds is available to beamline users
CASK functions as a Mg2+-independent neurexin kinase.
No full textSummaryCASK 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
High Throughput Tomography (HiTT) on EMBL beamline P14 on PETRA III
Full text linkHere, high-throughput tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A high-photon-flux undulator beamline is used to perform tomographic phase-contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beam-time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin-embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high-throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/50110000165
Development of Theoretical and Experimental Methods of Time-Resolved Laue Protein Cristallography
No full textEntwicklung theoretischer und experimenteller Methoden für die zeitaufgelöste Laue-Protein-Kristallographie
Full text linkNew theoretical, experimental and computational methods for time-resolved Laue crystallography of macromolecules have been developed. A solution to the energy-overlap problem in Laue diffraction is described that does not require redundancy in the measurements. The new method follows a Bayesian approach with multi-dimensional probability density functions. The intensity components of reflection multiplets are deconvoluted, and estimates of their precision are obtained. The Laue patterns are processed to their physically relevant wavelength-dependent resolution limit; no "soft-parameters" are involved. The power of the method is demonstrated by the test applications to bovine trypsin, sperm whale myoglobin and 2Mn-catalase from Thermus thermophilus. In the example of trypsin, the completeness at low and medium resolution as well as at very high resolution (1.4 e) is enhanced very substantially as compared to standard procedures; the "low-resolution hole" problem is practically solved. As a consequence, the contrast in electron density maps improves so far that they become comparable in quality to maps from monochromatic data of the same resolution. The new method is of interest for all types of Laue diffraction experiments, in particular for single-shot time resolved studies on short time scales. In a stroboscopic test study of ground-state CO myoglobin, the Bayesian method permitted to obtain the electron density maps that showed the correct ligand conformation at resolution 1.7 e. The resolution that was achieved using DORIS and the map contrast are higher as compared to the earlier experiments of this type that has been carried out at the third generation synchrotron source. The possibility to obtain interpretable maps in a single-shot Laue experiment is demonstrated for the example of cubic catalase. A limited-bandwidth Laue method using a graphite monochromator with 2.5% bandwidth has been developed and tested in study of lysozyme at ultra-high resolution (200 e are presented.Neue theoretische, experimentelle und rechnerische Methoden für die zeitaufgelöste Laue-Kristallographie von Makromolekülen wurden entwickelt. Eine Lösung für das Energieüberlappungsproblem der Laue-Diffraktion wird beschrieben, die keine Redundanz in den Messdaten erfordert. Die neue Methode nutzt Bayes-Statistik mit mehrdimensionalen Wahrscheinlichkeitsdichtefunktionen. Die Intensitätskomponenten der Reflexmultiplets werden entfaltet und ihre Genauigkeit abgeschätzt. Die Laue-Beugungsbilder werden bis zur jeweiligen physikalisch relevanten wellenlängenabhängigen Auflösungsgrenze bearbeitet, ohne dabei "Soft parameter" anzunehmen. Die Leistungsfähigkeit der Methode wird anhand von Testanwendungen an Rindertrypsin, Pottwal-Myoglobin und 2Mn-Katalase des Thermus thermophilus demonstriert. Im Fall von Trypsin liegt die Vollständigkeit bei niedrigen und mittleren sowie bei sehr hohen Auflösungen (1.4 e) im Vergleich zu Laue-Standardmethoden um ein Vielfaches höher. Das Problem der geringen Vollständigkeit im Breich niederer Auflösung ("low-resolution-hole") ist praktisch gelöst. Als Folge erhöht sich der Kontrast der Elektronendichtekarten so weit, dass sie in ihrer Qualität mit Karten vergleichbar sind, die auf monochromatischen Daten gleicher Auflösung beruhen. Die neue Methode ist für alle Arten von Laue-Beugungsexperimenten, insbesonders auch für zeitaufgelöste Studien aufgrund einer einzigen Aufnahme (single shot) in kurzem Zeitmaßstab von Interesse. In einer stroboskopischen Teststudie an CO-Myoglobin im Grundzustand ermöglichte es die Bayes-Methode, Elektronendichtekarten zu erhalten, die die korrekte Konformation der Liganden bei einer Auflösung von 1.7e zeigten. Die Auflösung und der Kontrast der Dichtekarten, die aufgrund von Messungen an DORIS erhalten wurden, sind höher als bei früheren Experimenten dieser Art, obwohl diese an Strahlungsquellen der dritten Generation durchgeführt wurden. Die Möglichkeit interpretierbare Karten mit Single-Shot-Laue-Experimenten zu erhalten, wird am Beispiel von kubischer Katalase demonstriert. Zudem wurde eine Laue-Methode mit reduzierter Bandbreite (2.5%) unter Verwendung eines Graphit-Monochromators entwickelt und am Beispiel von Lysozym bei extrem hoher Auflösung (< 1.2 e) getestet. Schließlich wurde ein neuer Algorithmus zur Autoindizierung von Laue-Beugungsmustern entwickelt und in einem Computerprogramm implementiert. Erfolgreiche Anwendungen dieses Verfahrens auf Strukturen mit vergleichsweise großen Zellkonstanten werden vorgestellt
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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