Search all library resources

Structural dynamics of G protein-coupled receptor activation [electronic resource]

G protein-coupled receptors (GPCRs) are transmembrane proteins that mediate complex cellular responses to hormones and neurotransmitters. GPCRs can activate many signaling pathways, either through interactions with heterotrimeric G proteins or through G protein-independent pathways. The function of GPCRs can be modulated by a variety of ligands with a broad range of efficacies, often in a signaling pathway specific manner. Understanding this complex signaling behavior requires insight into the molecular mechanisms of GPCR function. Recent advances in GPCR crystallography have elucidated the structure of a few prototypical receptors, rhodopsin and the β2 adrenergic receptor (β2AR), in both inactive and active conformations. Here, I describe my efforts to understand the structure and dynamics of GPCR activation. To extend the available model systems beyond rhodopsin and the β2AR, I describe the inactive-state structures of the μ and δ opioid receptors as well as a recently determined structure of an activated, agonist-bound μ opioid receptor. I also describe active state structures of the M2 muscarinic receptor and higher resolution structures of the activated β2AR bound its endogenous hormone adrenaline. Finally, I describe a set of studies utilizing 19F-fluorine nuclear magnetic resonance (NMR) and double electron-electron resonance spectroscopy to assess the conformational dynamics of β2AR activation. Together, these studies establish the conformational complexity associated with GPCR activation and how this structural plasticity is likely responsible for the broad versatility of GPCR signaling.

High-density grids for efficient data collection from multiple crystals [electronic resource].

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into theBlu-Ice/DCSSexperimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

Online OSTI

Goniometer-based femtosecond crystallography with X-ray free electron lasers [electronic resource].

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. With smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β₂-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

Online OSTI