Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The g-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergen

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.Peer reviewe

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

15 pags., 6 figs., 1 tab.With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.Financial support from the STC Program of the National Science Foundation through BioXFEL under agreement no. 1231306, NSF ABI Innovations award no. 1565180, and the National Institutes of Health award no. R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687.Peer reviewe

Preventing Bio-Bloopers and XFEL Follies: Best Practices from your Friendly Instrument Staff

Serial Femtosecond Crystallography (SFX) at X-ray Free electron Lasers (XFELs) is a relatively new field promising to deliver unparalleled spatial and temporal resolution on biological systems and there dynamics. Over the past decade, though, there have been a handful of results that have truly delivered on these promises. Why? SFX has many paradigm shifting techniques not seen in typical structural biology arenas, such as creating a concentrated slurry of microcrystals rather than a handful of loopable prisms worthy of a catalog photo. Then taking that slurry and high speed jetting them towards the vacuum or helium interation region to destroy less than 1% of your sample and waste the other 99. The literature is full of techniques and methods promising to cure what ails your experiment, yet as an instrument scientist will tell you –and a first author might admit after a few drinks at the conference happy hour—is that there are a lot more failures than the success we published, results may vary. We will walk through a best practices on how to prepare your sample and chose a sample delivery technique that will amerliorate your studies rather than undermine your hardwork and hopefully lead to better experimental planning and execution, inching you closer to that scientific goal and that call from Stockholm. This will be written in a more editorialized fashion and is meant to give the reader an idea of what to try or how they should be thinking. Welcome to SFX, now what

Kinetic analysis of acetylation-dependent Pb1 bromodomain-histone interactions

Stopped-flow fluorescence anisotropy was used to determine the kinetic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains are acetyllysine binding motifs found in many chromatin associated proteins. Individual bromodomains were derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin-remodeling complex that has six tandem bromodomains in the amino-terminal region. The average kon and koff values for the formation of high-affinity complexes are 275 M- 1 s- 1 and 0.41 × 10- 3 s- 1, respectively. The average kon and koff values for the formation of low-affinity complexes are 119 M- 1 s- 1 and 1.42 × 10- 3 s- 1, respectively. Analysis of the on- and off-rates yields acetylation site-dependent equilibrium dissociation constants averaging 1.4 and 12.9 μM for high- and low-affinity complexes, respectively. This work represents the first examination of kinetic mechanisms of acetylation-dependent bromodomain-histone interactions. © 2008 Elsevier B.V. All rights reserved

Versuchsaufbau zum Test und Evaluierung von Kalman Filter Konfigurationen

Die Positionierung mobiler Systeme mit hoher Genauigkeit ist eine Voraussetzung für intelligentes autonomes Verhalten, sowohl in der Feldrobotik als auch in industriellen Umgebungen. Dieser Beitrag beschreibt den Aufbau einer Roboterplattform und ihre Verwendung für den Test und die Bewertung von Kalman-Filter-Konfigurationen. Der Aufbau wurde mit einem mobilen Roboter Husky A200 und einem LiDAR-Sensor (Light Detection and Ranging) realisiert. Zur Verifizierung des vorgeschlagenen Aufbaus wurden fünf verschiedene Szenarien ausgearbeitet. Mit denen wurden die Filter auf ihre Leistungsfähigkeit hinsichtlich der Genauigkeit der Positionsbestimmung getestet.Positioning mobile systems with high accuracy is a prerequisite for intelligent autonomous behavior, both in field robotics and in industrial environments. This paper describes the setup of a robotic platform and its use for testing and evaluating Kalman filter configurations. The setup was implemented using a Husky A200 mobile robot and a light detection and ranging (LiDAR) sensor. Five different scenarios were devised to verify the proposed setup. With these, the filters were tested for their performance in terms of position determination accuracy

Evaluierung von Kalman Filter Konfigurationen zur Roboterlokaliserung mittels Sensordatenfusion

In dieser Arbeit werden drei verschiedene Konfigurationen der von Tom Moore, für das Robot Operating System, entwickelte Kalman-Filter vorgestellt. Diese bilden die Grundlage für eine Lokalisierung mittels Sensorfusion in dem verwendeten ROS-Framework. Ziel dieser Arbeit ist der Aufbau und die Verifikation einer Lokalisierung für ein mobiles Robotersystem Husky A200 der Firma Clearpath Robotics. Hierzu wurden die Möglichkeiten des bestehenden Systems untersucht und mehrere Versionen von Lokalisierungsfiltern konfiguriert. Am an Ende, wird eine Verifikation der Ergebnisse in verschiedenen Szenarien gegeneinandergestellt. Hierzu werden die Ergebnisse einer Variante des Extended Kalman-Filters in 2D (EKF2D), eine Variante des Unscented Kalman-Filter in 2D (UKF2D) und eine Variante des Extended Kalman-Filters in 3D (EKF3D) verifiziert und verglichen. Die Untersuchungen ergaben das der EKF2D die besten und robustesten Ergebnisse für eine Lokalisierung erbringt, trotz, im Vergleich zu der UKF2D Variante, 17,3 % höhere Endpositionsabweichung aufweist. Die in diesem Projekt gewählte EKF3D Konfigurationsvariante eignet sich, wegen seinen starken Ungenauigkeiten in der Höhenbestimmung nicht für eine aussagekräftige Positionsbestimmung.In this work, three different configurations of the Kalman filter developed by Tom Moore for the Robot Operating System are presented. These form the basis for localization using sensor fusion in the ROS framework used. The aim of this work is the construction and verification of localization for a mobile robot system Husky A200 from Clearpath Robotics. For this purpose, the possibilities of the existing system were examined, and several versions of localization filters were configured. In the end, a verification of the results in different scenarios is compared. For this purpose, the results of a variant of the Extended Kalman filter in 2D (EKF2D), a variant of the Unscented Kalman filter in 2D (UKF2D), and a variant of the Extended Kalman filter in 3D (EKF3D) are verified and compared. The investigations showed that the EKF2D provides the best and most robust results for localization, despite having a 17.3% higher end position deviation compared to the UKF2D variant. The EKF3D configuration variant selected in this project is not suitable for a meaningful position determination due to its strong inaccuracies in height determination