自由电子激光在生物学中的应用

同步辐射的发展和应用已经极大的推动了自然科学包括生物学的巨大发展,其中结构生物学更是离不开X射线衍射分析,小角散射等。X射线自由电子激光(XFEL)相比同步辐射具有更高强度,完全相干等特点,被称为第四代光源。科学家已经利用XFEL实现了尺度约为1微米的蛋白质晶体的高分辨率结构解析,并且也实现了单颗粒的病毒的低分辨重构。未来,XFEL将会为生物学的发展打开一扇新的大门。     

X-ray structure determination of the glycine cleavage system protein H of Mycobacterium tuberculosis using an inverse Compton synchrotron X-ray source

Structural genomics discovery projects require ready access to both X-ray diffraction and NMR spectroscopy which support the collection of experimental data needed to solve large numbers of novel protein structures. The most productive X-ray crystal structure determination laboratories make extensive use of tunable synchrotron X-ray light to solve novel structures by anomalous diffraction methods. This requires that frozen cryo-protected crystals be shipped to large multi acre synchrotron facilities for data collection. In this paper we report on the development and use of the first laboratory-scale synchrotron light source capable of performing many of the state-of-the-art synchrotron applications in X-ray science. This Compact Light Source is a first-in-class device that uses inverse Compton scattering to generate X-rays of sufficient flux, tunable wavelength and beam size to allow high-resolution X-ray diffraction data collection from protein crystals. We report on benchmarking tests of X-ray diffraction data collection with hen egg white lysozyme, and the successful high-resolution X-ray structure determination of the Glycine cleavage system protein H from Mycobacterium tuberculosis using diffraction data collected with the Compact Light Source X-ray beam.     

Stopped-flow solution scattering using synchrotron radiation: Apparatus,data collection and data analysis

We have constructed an experimental system, under remote control, for stopped-flow X-ray scattering using synchrotron radiation. It has been used, in conjunction with an annular detector and its associated electronics, to obtain good scattering curves, with time-slices as short as 200 ms, in a new study of the dissociation of the enzyme complex aspartate transcarbamylase. The data have been analysed by new statistical methods, and they agree well with the results from parallel chemical quench experiments. For studying dissociation reactions, stopped-flow X-ray scattering is a quite practical method, which need not use very much more material than conventional stopped-flow experiments.     

Structure of tumour necrosis factor by X-ray solution scattering and preliminary studies by single crystal X-ray diffraction

The structure of tumour necrosis factor has been investigated by X-ray small-angle scattering and X-ray diffraction using synchrotron radiation. The overall radius of gyration is 25.5 A. A plausible model accounting for the scattering curves consists of an elongated trimer with an axial ratio of 3 to 4 and a maximal chord with a lower limit of 80 A. Tumour necrosis factor has been crystallized in a trigonal space group. Our results are in favour of a single trimer in the asymmetric unit. The diffraction extends to 3.5 A.     

The beamlines of ELETTRA and their application to structural biology

Protein crystallographers are nowadays regular users of synchrotron radiation (SR) facilities for several applications. The goal of majority of users is simply to extract more accurate, higher resolution data from existing crystals; they use monochromatic radiation and the rotation method, in order to get a complete survey of the reciprocal space in a short time. In fact the brilliance of SR is essential, due to the weak scattering power of the samples, and because of their sensibility to radiation damage. Over the last few years, however, a general increase of interest for measurements at multiple wavelengths, which exploit the anomalous dispersion for the phase problem (multiwavelength anomalous diffraction — MAD), has generated the need of intense tuneable sources. For these applications, the emphasis is on accurate measurements of the small differences between the intensities of Bragg reflections at various energies across the absorption edge of an element present in the sample. The macromolecular diffraction beamline at ELETTRA, which is now running routinely since spring 1995, has been designed to provide a high flux — highly collimated tuneable X-rays source in the spectral range between 4 and 25 keV. The radiation source is the 57-pole wiggler, which delivers a very intense radiation up to 25 keV, and is shared and used simultaneously with the small angle X-ray scattering (SAXS) beamline. The front-end filter system has a cut-off energy at about 4 keV. The beamline optics consists of a pseudo-channel-cut double-crystal monochromator followed by a double focusing toroidal mirror. The tunability and the stability of the monochromator allows the user to perform MAD experiments, and for this purpose, a fluorescence probe for the exact calibration of the absorption edge is available on-line. The experimental station is based on an imaging plate area detector from MarResearch, with a sensible area of 345 mm in diameter. A cooled N₂-stream is available to cool the sample crystal in order to reduce the radiation damage. SAXS is an experimental technique used to derive structural information about supra-molecular assemblies, amorphous materials and partly ordered systems (e.g. size and shape of large molecules). The high-flux SAXS beamline at ELETTRA is mainly intended for time-resolved studies on fast structural transitions in the sub-millisecond time region in solutions and in partly ordered systems, triggered by external or process parameters, with a SAXS resolution between 10 and 1400 Å in real space. The source is the already mentioned 57-pole and the SAXS beamline accepts three discrete energies of its spectrum, namely 5.4, 8 and 16 keV. The beamline optics consists of a flat double-crystal monochromator and a double focusing toroidal mirror. A multi-purpose sample stage, movable along an optical table in order to optimise the sample to detect distance, allows to perform fast time-resolved relaxation studies based on temperature- or pressure-jumps as well as stopped flow experiments. Moreover, the users have option to install their own specialised sample surrounding equipment. The optimisation of the beamline with respect to high-flux and consequently high-flux density, allows to perform the following experiments: low contrast solution scattering, grazing incidence surface diffraction, micro-spot scanning, X-ray fluorescence analysis, time-resolved studies 11 s, simultaneous small- and wide-angle measurements on gels, liquid crystals, biopolymers, amorphous materials, muscles.This revised version was published online in October 2005 with corrections to the Cover Date.     

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.     

Experience in the use of synchrotron radiation for the x-ray study of biopolymers]

The results of methodical work, carried out on the sources of synchrotron radiation (SR) with the aim of using SR as a powerful source of X-rays for studying biopolymer structure, are presented. The questions of monochromatization are considered. The technique designed for photoregistration of diffraction patterns within the wide range of scattering angles is described. X-ray diffraction patterns of feather ceratin, collagen and striated muscle are obtained with exposure periods ten times less than those in the case of X-ray tubes. The high resolution of diffraction lines, the absence of parasitic phone and the presence of reflections within the wide range of scattering angles are characteristic of these patterns.     

Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques

Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g. changes in redox states and bond lengths). In situ experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ experiments (e.g. on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ and in situ samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.     

Opportunities and challenges for time-resolved studies of protein structural dynamics at X-ray free-electron lasers

X-ray free-electron lasers (XFELs) are revolutionary X-ray sources. Their time structure, providing X-ray pulses of a few tens of femtoseconds in duration; and their extreme peak brilliance, delivering approximately 10¹² X-ray photons per pulse and facilitating sub-micrometre focusing, distinguish XFEL sources from synchrotron radiation. In this opinion piece, I argue that these properties of XFEL radiation will facilitate new discoveries in life science. I reason that time-resolved serial femtosecond crystallography and time-resolved wide angle X-ray scattering are promising areas of scientific investigation that will be advanced by XFEL capabilities, allowing new scientific questions to be addressed that are not accessible using established methods at storage ring facilities. These questions include visualizing ultrafast protein structural dynamics on the femtosecond to picosecond time-scale, as well as time-resolved diffraction studies of non-cyclic reactions. I argue that these emerging opportunities will stimulate a renaissance of interest in time-resolved structural biochemistry.     

Energy dispersive X-ray diffraction by collagen fibrils in costal cartilage using synchrotron radiation

Low-angle X-ray diffraction patterns from the isotropic distribution of collagen fibrils, which occur in low concentrations in costal cartilage, were recorded using synchrotron radiation. An energy dispersive technique was used to exploit the properties of synchrotron radiation to the full. The third, fourth, fifth and sixth diffraction orders from the axial periodicity of the fibrils were recorded and used to calculate a value for this periodicity of 67 ± 1 nm. This result is in good agreement with measurements made on amianthoid areas as well as from fibrils in tendon, which consist of a chemically distinct form of collagen.     

小角X射线散射实验中蛋白质溶液的辐照损伤及防护方法

随着同步辐射光源（尤其是目前快速发展的第四代同步辐射光源）技术的进步,可用于实验的辐射通量越来越高,实验样品（特别是蛋白质等生物大分子样品）受到的辐照损伤也越来越严重。在全球现有的同步辐射装置上,蛋白质等生物大分子溶液专用小角X射线散射（SAXS）实验站的光子通量基本上都在1013cps量级。在如此高的通量下,蛋白质等生物大分子溶液样品在实验测量中受到的辐照损伤极其严重。如果没有有效的辐照防护措施,蛋白质溶液样品在毫秒级辐照时间内便会辐照损伤,导致不能获取有效的实验数据。辐照损伤严重制约了SAXS实验技术在蛋白质溶液样品方面的应用。因而,认识蛋白质溶液样品辐照损伤的产生机理、影响因素、判断标准,以及有效降低辐照损伤程度、延缓辐照损伤产生时间的方法,对于蛋白质等生物大分子溶液的散射实验具有重要的指导意义。本文在简要概述生物大分子溶液样品辐照损伤产生机理、影响因素、辐照剂量等基本概念的基础上,重点综述了同步辐射SAXS实验中辐照损伤的判断标准和防护措施。此外,本文还对比了各种防护措施的优缺点,讨论了在建HEPS新光源中SAXS束线可用的散射数据采集时间,指出辐照损伤防护剂是有价值的研究方向...     

Biological applications of synchrotron radiation infrared spectromicroscopy

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (< 10 μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively.     

Physicochemical Characterization of the Reassembled Dimer of an Integral Membrane Protein OmpF Porin

The in vitro reassembled species of OmpF porin, which was renatured from its denatured monomer using n-octyl-β-D-glucopyranoside, was characterized by low-angle laser light scattering photometry, circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering measurements. The light scattering measurement reconfirmed that the reassembled species was the dimer of the protein. Circular dichroism spectra of the reassembled dimer showed a native-like β-structure. A small-angle X-ray scattering measurement indicated that the size of the reassembled dimer was nearly equal to that of the native trimer under the present experimental conditions. In a thermal denaturation experiment followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the reassembled dimer was less stable than the native trimer.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

Lipid discrimination in phospholipid monolayers by the antimicrobial frog skin peptide PGLa. A synchrotron X-ray grazing incidence and reflectivity study

We present a first study using synchrotron grazing incidence diffraction and X-ray reflectivity measurements on mixed phospholipid/peptide monolayers at the air/water interface. The thermodynamic properties of the pure and mixed monolayers were characterized using the classical film balance technique. Surface pressure/potential-area isotherms showed that the antimicrobial frog skin peptide PGLa formed a very stable monolayer with two PGLa molecules per kinetic unit and a collapse pressure of ~22 mN/m. X-ray grazing incidence diffraction indicated that the peptide-dimer formation did not lead to self-aggregation with subsequent crystallite formation. However, the scattering length density profiles derived from X-ray reflectivity measurements yield information on the PGLa monolayer that protrudes into the air phase by about 0.8 nm, suggesting that the peptide is aligned parallel to the air/water interface. The monolayers, composed of disaturated phosphatidylcholines or phosphatidylglycerols, were stable up to 60 mN/m and exhibited a first-order transition from a liquid-expanded to a liquid-condensed state around 10 mN/m. Structural details of the phospholipid monolayers in the presence and absence of PGLa were obtained from synchrotron experiments. Thereby, the X-ray data of distearoylphosphatidylcholine/PGLa can be analyzed by being composed of the individual components, while the peptide strongly perturbed the lipid acyl chain order of distearoylphosphatidylglycerol. These results are in agreement that PGLa mixes at a molecular level with negatively charged lipids, but forms separate islands in zwitterionic phosphatidylcholine monolayers and demonstrates that antimicrobial peptides can discriminate between the major phospholipid components of bacterial and mammalian cytoplasmic membranes.     

The location of redox centers in biological membranes determined by resonance x-ray diffraction. I. Observation of the resonance effect

We have developed resonance X-ray diffraction methods to locate for the first time intrinsic metal atoms associated with redox centers within biological membrane systems. The study of membranes containing dilute concentrations of resonant scatterers has been made possible by the development of synchrotron radiation sources of X-rays. The technique permits altering the scattering power of a particular atom relative to others by varying the incident X-ray energy. Thus, this method may be used to locate a metal atom within a complex integral protein without chemical modification of the membrane. We present resonance diffraction data taken with synchroton radiation for two different membrane systems: cytochrome oxidase incorporated into lipid vesicles and a photosynthetic reaction center-cytochrome c complex also reincorporated into lipid vesicles.     

New techniques in macromolecular cryocrystallography: macromolecular crystal annealing and cryogenic helium

Cryocrystallography is used today for almost all X-ray diffraction data collection at synchrotron beam lines, with rotating-anode generators, and micro X-ray sources. Despite the widespread use of flash-cooling to place macromolecular crystals in the cryogenic state, its use can ruin crystals, trips to the synchrotron, and sometimes even an entire project. Annealing of macromolecular crystals takes little time, requires no specialized equipment, and can save crystallographic projects that might otherwise end in failure. Annealing should be tried whenever initial flash-cooling causes an unacceptable increase in mosaicity, results in ice rings, fails to provide adequate diffraction quality, or causes a crystal to be positioned awkwardly. Overall, annealing improves the quality of data and overall success rate at synchrotron beam lines. Its use should be considered whenever problems arise with a flash-cooled crystal. Helium is a more efficient cryogen than nitrogen and will deliver lower temperatures. Experiments suggest that when crystals are cooled with He rather than N2, crystals maintain order and high-resolution data are less affected by increased radiation load. Individually or in combination, these two techniques can enhance the success of crystallographic data collection, and their use should be considered essential for high-throughput programs.     

Functional lung imaging with synchrotron radiation: Methods and preclinical applications

Many lung disease processes are characterized by structural and functional heterogeneity that is not directly appreciable with traditional physiological measurements. Experimental methods and lung function modeling to study regional lung function are crucial for better understanding of disease mechanisms and for targeting treatment. Synchrotron radiation offers useful properties to this end: coherence, utilized in phase-contrast imaging, and high flux and a wide energy spectrum which allow the selection of very narrow energy bands of radiation, thus allowing imaging at very specific energies. K-edge subtraction imaging (KES) has thus been developed at synchrotrons for both human and small animal imaging. The unique properties of synchrotron radiation extend X-ray computed tomography (CT) capabilities to quantitatively assess lung morphology, and also to map regional lung ventilation, perfusion, inflammation and biomechanical properties, with microscopic spatial resolution. Four-dimensional imaging, allows the investigation of the dynamics of regional lung functional parameters simultaneously with structural deformation of the lung as a function of time. This review summarizes synchrotron radiation imaging methods and overviews examples of its application in the study of disease mechanisms in preclinical animal models, as well as the potential for clinical translation both through the knowledge gained using these techniques and transfer of imaging technology to laboratory X-ray sources.     

Novel methods for studying lipids and lipases and their mutual interaction at interfaces. Part II. Surface sensitive synchrotron X-ray scattering

Monolayers of lipids have been studied for more than a century. During the past decade new insight into the field has resulted from the development of surface sensitive X-ray scattering methods utilizing synchrotron radiation: grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). These novel methods provide direct microscopic information about the systems in question and allow in situ investigations under near physiological conditions. GIXD gives information about the in-plane molecular structure, e.g., lattice symmetry and structural parameters; XR provides the electron density profile across the interface. The present review describes the theory, experimental procedures and sample requirements for surface sensitive X-ray scattering. An overview of recent results is presented as well, with special emphasis on biologically important systems, e.g., investigations by GIXD and/or XR of lipid and protein structures at interfaces and of lipid/protein interactions.     

Proteins in action: the physics of structural fluctuations and conformational changes

Structural dynamics is essential for the biological function of proteins. Results from new experimental techniques should be compared with those from previous experiments in order to obtain a consistent picture of the physics of intramolecular fluctuations and conformational changes. The high intensity and time structure of synchrotron radiation have made possible time-resolved X-ray structure analysis and the determination of phonon density spectra through the M?ssbauer effect. By combining results from M?ssbauer absorption spectroscopy, incoherent neutron scattering, low-temperature crystallography and optical spectroscopy, a physical picture of protein dynamics emerges.