Daniel J. Thiel

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex

BACKGROUND Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

Determination of a protein structure by iodination: the structure of iodinated acetylxylan esterase.

Enzymatic and non-enzymatic iodination of the amino acid tyrosine is a well known phenomenon. The iodination technique has been widely used for labeling proteins. Using high-resolution X-ray crystallographic techniques, the chemical and three-dimensional structures of iodotyrosines formed by non-enzymatic incorporation of I atoms into tyrosine residues of a crystalline protein are described. Acetylxylan esterase (AXE II; 207 amino-acid residues) from Penicillium purpurogenum has substrate specificities towards acetate esters of D-xylopyranose residues in xylan and belongs to a new class of alpha/beta hydrolases. The crystals of the enzyme are highly ordered, tightly packed and diffract to better than sub-angström resolution at 85 K. The iodination technique has been utilized to prepare an isomorphous derivative of the AXE II crystal. The structure of the enzyme determined at 1.10 A resolution exclusively by normal and anomalous scattering from I atoms, along with the structure of the iodinated complex at 1.80 A resolution, demonstrate the formation of covalent bonds between I atoms and C atoms at ortho positions to the hydroxyl groups of two tyrosyl moieties, yielding iodotyrosines.

Submicron concentration and confinement of hard X-rays

Abstract High-intensity hard X-ray beams of submicron diameters were generated using a tapered glass capillary as a concentrator. A complete description of the fabrication and characterization of such capillary optics is given. A new method for producing submicron X-ray collimators is also described.

Microbeam generation with capillary optics (invited)

Grazing incidence x‐ray optics for microbeam generation can be classified into five types: ellipsoidal mirror, Wolter mirror, monocapillary concentrator, microchannel array, and polycapillary concentrator. These components each have their own properties, yet they are closely related. Each optical component is at a different stage of development. Ellipsoidal mirrors are based on a mature technology and at 1/10 magnification should yield 10‐μm‐diam beams. Optics based on replicate Wolter mirrors are capable of producing beams on a 1–10 μm scale with high gain. Monocapillary concentrators are producing beam sizes of less than 0.1 μm. On a larger scale, polycapillary concentrators, and microchannel arrays are promising microbeam components. Ray tracing programs exist in different forms for some of these components. Prototype capillary optics have been tested, but as a whole, the manufacturing methods could be significantly improved with further investments in time and effort. All of these optical designs show...

Focusing of synchrotron radiation using tapered glass capillaries

Abstract A simple technique for focusing synchrotron radiation and thereby providinghigh intensity x-ray beams as small as 0.1 micron in diameter is described. Gradually tapered glass capillaries serve as the focusing elements. The first measurements of gain in the intensity of x-rays using focusing capillaries have been made.

Production of intense micrometer‐sized x‐ray beams with tapered glass monocapillaries

Methods were developed to characterize tapered capillaries as x‐ray concentrators capable of forming spots of intense x‐ray radiation with micrometer diameters. These tapered capillaries, with somewhat controlled tapers, were produced using a gravity‐based capillary puller. A device was constructed to microscopically inspect these capillaries along two orthogonal axes in order to accurately measure the tapering and bending. Both monochromatic and white hard x rays were concentrated with a variety of tapered capillaries, and the subsequent gains in intensity (flux/area) ranging from 14 to 35 are reported. Using these unique x‐ray concentrators, a simple high‐powered x‐ray fluorescence microscope was constructed and tested. We also found that hard x‐ray beams could be successfully steered by bending the capillary tip with radii as small as 5 m. In addition, preliminary ray‐tracing results obtained from a two‐dimensional ray‐tracing program are described.

Guiding and concentrating hard x-rays by using a flexible hollow-core tapered glass fiber

A 1.6-m-long, flexible, hollow glass fiber with a gradually diminishing bore diameter has been used efficiently to compress the size of an x-ray beam as it reflects from the inside walls of the fiber by total external reflection. The transmission characteristics of the fiber are reported for monochromatic synchrotron radiation of 8.04, 13, and 20 keV, as well as for CuKalpha radiation from a conventional x-ray tube. Intensity enhancements as large as 10 that correspond to a transmission efficiency of 54% were observed. The high efficiency of this prototype fiber supports the idea that this confinement technique should yield intensity gains of many orders of magnitude as the optimal fiber design is achieved.

High-resolution macromolecular structure determination using CCD detectors and synchrotron radiation

BACKGROUND Synchrotron radiation sources have made impressive contributions to macromolecular crystallography. The delay in development of appropriate X-ray detectors has, however, been a significant limitation to their efficient use. New technologies, based on charge-coupled devices (CCDs), provide capabilities for faster, more accurate, automated data collection. RESULTS A CCD-based X-ray detector has been developed for use in macromolecular crystallography and has been in operation for about one and a half years at the Cornell High Energy Synchrotron Source. It has been used for a variety of crystallographic projects, including a number of high-resolution structural studies. The statistical quality of the data, the detectors ease and efficiency of use, and the growing number of structural results illustrate the practical utility of this new detector system. CONCLUSIONS The new detector has enhanced capabilities for measuring diffraction patterns from crystals of macromolecules, especially at high resolution, when the X-ray intensities are weak. The survey of results described here ranges from virus crystallography to weakly diffracting small-molecule structure determination and demonstrates the potential of CCD detectors when combined with synchrotron radiation sources.

Developments in tapered monocapillary and polycapillary glass X-ray concentrators

Abstract Tapered glass capillaries can be used to condense both wide-band and monochromatic hard (> 5 keV) X-ray beams. These long, hollow, needle-like optics have been used in a variety of experiments to demonstrate their utility in areas such as protein crystallography, hard X-ray imaging, and Laue diffraction. Monocapillary versions of these X-ray concentrators have yielded intensity gains of up to 1000 and X-ray beam sizes as small as 50 nm. Recently, in an effort to make larger condensed beam sizes (> 10 μ m), polycapillary tubing has been tapered and a 68 μm diameter X-ray beam with an intensity gain of 4.6 was observed using 6 keV synchrotron radiation. Results from developments of both types of tapered capillaries and from ray-tracing design efforts are presented.

Focussing optics for a synchrotron-based X-ray microprobe

Abstract X-ray microprobe experiments at third-generation synchrotron sources will provide trace element analysis of samples with 1 μm × 1 μm spatial resolution and femtogram sensitivity. For these experiments to be possible. X-ray optical elements need to be developed to focus the beam from a hard X-ray undulator to a micron spot size. In June 1991 several different optical elements were tested during a dedicated undulator run at CHESS. The undulator produced radiation similar to that which will be available at third-generation sources like the Advanced Photon Source. Both Fresnel zone plates and multilayer-coated spherical mirrors were tested. With the Fresnel zone plate a spot size of 8.5 μm × 30 μm was achieved in the first order and 6 μm × 20 μm in the second order. With a Kirkpatrick-Baez multilayer mirror system a spot size of 4 μm × 9 μm was achieved. Based on these results, some of the requirements for an optical system suitable for a dedicated microprobe beamline are given.