Robert F. Fischetti

Crystal structure of the human beta2 adrenergic G-protein-coupled receptor.

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human β2 adrenoceptor (β2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å/3.7 Å resolution. The cytoplasmic ends of the β2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the β2AR are not seen. The β2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the β2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

Crystal structure of the human β2 adrenergic G-protein-coupled receptor

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human β2 adrenoceptor (β2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å/3.7 Å resolution. The cytoplasmic ends of the β2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the β2AR are not seen. The β2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the β2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

Crystal structure of the human |[bgr]|2 adrenergic G-protein-coupled receptor

Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human β2 adrenoceptor (β2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 Å/3.7 Å resolution. The cytoplasmic ends of the β2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the β2AR are not seen. The β2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the β2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

The myosin motor in muscle generates a smaller and slower working stroke at higher load

Muscle contraction is driven by the motor protein myosin II, which binds transiently to an actin filament, generates a unitary filament displacement or ‘working stroke’, then detaches and repeats the cycle. The stroke size has been measured previously using isolated myosin II molecules at low load, with rather variable results, but not at the higher loads that the motor works against during muscle contraction. Here we used a novel X-ray-interference technique to measure the working stroke of myosin II at constant load in an intact muscle cell, preserving the native structure and function of the motor. We show that the stroke is smaller and slower at higher load. The stroke size at low load is likely to be set by a structural limit; at higher loads, the motor detaches from actin before reaching this limit. The load dependence of the myosin II stroke is the primary molecular determinant of the mechanical performance and efficiency of skeletal muscle.

Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 µm size X-ray synchrotron beam

Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 µm minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets.

Micro-crystallography comes of age

The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beam lines. Brilliant X-ray beams of diameter 20 μm or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography.

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.

A 7 µm mini-beam improves diffraction data from small or imperfect crystals of macromolecules

An X-ray mini-beam of 8 × 6 µm cross-section was used to collect diffraction data from protein microcrystals with volumes as small as 150–300 µm3. The benefits of the mini-beam for experiments with small crystals and with large inhomogeneous crystals are investigated.

High-resolution wide-angle X-ray scattering of protein solutions: effect of beam dose on protein integrity

Wide-angle X-ray scattering patterns from proteins in solution contain information relevant to the determination of protein fold. At relevant scattering angles, however, these data are weak, and the degree to which they might be used to categorize the fold of a protein is unknown. Preliminary work has been performed at the BioCAT insertion-device beamline at the Advanced Photon Source which demonstrates that one can collect X-ray scattering data from proteins in solution to spacings of at least 2.2 A (q = 2.8 A(-1)). These data are sensitive to protein conformational states, and are in good agreement with the scattering predicted by the program CRYSOL using the known three-dimensional atomic coordinates of the protein. An important issue in the exploitation of this technique as a tool for structural genomics is the extent to which the high intensity of X-rays available at third-generation synchrotron sources chemically or structurally damage proteins. Various data-collection protocols have been investigated demonstrating conditions under which structural degradation of even sensitive proteins can be minimized, making this technique a viable tool for protein fold categorization, the study of protein folding, unfolding, protein-ligand interactions and domain movement.

JBluIce–EPICS control system for macromolecular crystallography

The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.