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One of the brilliant ideas of John Spence when he saw the first diffraction patterns from the Linac Coherent Light Source was that one could solve the crystallographic phase problem by utilising the intensities between Bragg peaks. Because these intensities are due to the Fourier transform of the shape of the crystal, the approach came to be known as "shape-transform phasing." Shape-transform phasing was developed over the next ten years and formed the basis for many other interesting ideas and pursuits. Here we describe the current best implementation of the original idea using a lattice occupancy formalism and show that certain types of crystal defects can also be modelled via this approach, allowing the molecular structure to be recovered from the additional information offered by the inter-Bragg intensities from these crystal defects.
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Achromatic focusing systems for hard X-rays are examined which consist of a refractive lens paired with a diffractive lens. Compared with previous analyses, we take into account the behaviour of thick refractive lenses, such as compound refractive lenses and waveguide gradient index refractive lenses, in which both the focal length and the position of the principal planes vary with wavelength. Achromatic systems formed by the combination of such a thick refractive lens with a multilayer Laue lens are found that can operate at a focusing resolution of about 3 nm, over a relative bandwidth of about 1%. With the appropriate distance between the refractive and diffractive lenses, apochromatic systems can also be found, which operate over relative bandwidth greater than 10%. These systems can be used to focus short pulses without distorting them in time by more than several attoseconds. Such systems are suitable for high-flux scanning microscopy and for creating high intensities from attosecond X-ray pulses.
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X-ray microscopy offers the opportunity to image biological and radiosensitive materials without special sample preparations, bridging optical and electron microscopy capabilities. However, the performance of such microscopes, when imaging radiosensitive samples, is not limited by their intrinsic resolution, but by the radiation damage induced on such samples. Here, we demonstrate a novel, to the best of our knowledge, radio-efficient microscope, scanning Compton X-ray microscopy (SCXM), which uses coherently and incoherently (Compton) scattered photons to minimize the deposited energy per unit of mass for a given imaging signal. We implemented SCXM, using lenses capable of efficiently focusing 60 keV X-ray photons into the sub-micrometer scale, and probe its radio-efficient capabilities. SCXM, when implemented in high-energy diffraction-limited storage rings, e.g., European Synchrotron Radiation Facility Extremely Brilliant Source and PETRA IV, will open the opportunity to explore the nanoscale of unstained, unsectioned, and undamaged radiosensitive materials.
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Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.
Assuntos
Coenzimas/química , Cristalização , IMP Desidrogenase/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Guanosina Monofosfato , Modelos Moleculares , Conformação Proteica , Células Sf9 , Trypanosoma brucei brucei/genéticaRESUMO
We introduce a way to stabilize steady micro/nanoliquid jets issuing from Taylor cones together with coflowing gas streams. We study the dripping-jetting transition of this configuration theoretically through a global stability analysis as a function of the governing parameters involved. A balance between the local radial acceleration to the surface tension gradient, the mass conservation, and the energy balance equations enable us to derive two coupled scaling laws that predict both the minimum jet diameter and its maximum velocity. The theoretical prediction provides a single curve that describes not only the numerical computations but also experimental data from the literature for cone jets assisted with gas coflow. Additionally, we performed a set of experiments to verify what parameters influence the jet length. We adopt a very recent model for capillary jet length to our configuration by combining electrohydrodynamic effects with the gas flow through an equivalent liquid pressure. Due to diameters below 1 µm and high speeds attainable in excess of 100 m/s, this concept has the potential to be utilized for structural biology analyses with x-ray free-electron lasers at megahertz repetition rates as well as other applications.
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Reliable sample delivery and efficient use of limited beam time have remained bottlenecks for serial crystallography (SX). Using a high-intensity polychromatic X-ray beam in combination with a newly developed charge-integrating JUNGFRAU detector, we have applied the method of fixed-target SX to collect data at a rate of 1â kHz at a synchrotron-radiation facility. According to our data analysis for the given experimental conditions, only about 3 000 diffraction patterns are required for a high-quality diffraction dataset. With indexing rates of up to 25%, recording of such a dataset takes less than 30â s.
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During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9â Å resolution using automatic model-building tools.
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The entatic state denotes a distorted coordination geometry of a complex from its typical arrangement that generates an improvement to its function. The entatic-state principle has been observed to apply to copper electron-transfer proteins and it results in a lowering of the reorganization energy of the electron-transfer process. It is thus crucial for a multitude of biochemical processes, but its importance to photoactive complexes is unexplored. Here we study a copper complex-with a specifically designed constraining ligand geometry-that exhibits metal-to-ligand charge-transfer state lifetimes that are very short. The guanidine-quinoline ligand used here acts on the bis(chelated) copper(I) centre, allowing only small structural changes after photoexcitation that result in very fast structural dynamics. The data were collected using a multimethod approach that featured time-resolved ultraviolet-visible, infrared and X-ray absorption and optical emission spectroscopy. Through supporting density functional calculations, we deliver a detailed picture of the structural dynamics in the picosecond-to-nanosecond time range.
Assuntos
Complexos de Coordenação/química , Cobre/química , Processos Fotoquímicos , Teoria da Densidade Funcional , Transporte de Elétrons , Estrutura MolecularRESUMO
Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.
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Cristalografia por Raios X/métodos , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/estatística & dados numéricos , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Endopeptidase K/química , Desenho de Equipamento , Modelos Moleculares , Ficocianina/química , Conformação Proteica , Eletricidade Estática , Síncrotrons , Difração de Raios XRESUMO
Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.
Assuntos
Cristalografia por Raios X/métodos , Nanotecnologia/métodos , Conformação de Ácido Nucleico , RNA Bacteriano/química , Riboswitch , Regiões 5' não Traduzidas/genética , Aptâmeros de Nucleotídeos/química , Cristalização , Difusão , Elétrons , Cinética , Lasers , Ligantes , Modelos Moleculares , Dobramento de RNA , RNA Bacteriano/genética , Fatores de Tempo , Vibrio vulnificus/genéticaRESUMO
X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven atto-second X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser-, accelerator,- X-ray scientists as well as spectroscopists and biochemists optimizes X-ray pulse parameters, in tandem with sample delivery, crystal size, and advanced X-ray detectors. Ultimately, the new capability, attosecond serial X-ray crystallography and spectroscopy, will be applied to one of the most important problems in structural biology, which is to elucidate the dynamics of light reactions, electron transfer and protein structure in photosynthesis.
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Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
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Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today's third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the goal of high-throughput structure determination with X-ray FELs.
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A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 µm particles can be focused to a full-width at half maximum diameter of 4.2 µm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.
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Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.
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We give a detailed account of the theoretical analysis and the experimental results of an X-ray-diffraction experiment on quantum-state selected and strongly laser-aligned gas-phase ensembles of the prototypical large asymmetric rotor molecule 2,5-diiodobenzonitrile, performed at the Linac Coherent Light Source [Phys. Rev. Lett.112, 083002 (2014)]. This experiment is the first step toward coherent diffractive imaging of structures and structural dynamics of isolated molecules at atomic resolution, i.e., picometers and femtoseconds, using X-ray free-electron lasers.
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The structures of biological molecules may soon be determined with X-ray free-electron lasers without crystallization by recording the coherent diffraction patterns of many identical copies of a molecule. Most analysis methods require a measurement of each molecule individually. However, current injection methods deliver particles to the X-ray beam stochastically and the maximum yield of single particle measurements is 37% at optimal concentration. The remaining 63% of pulses intercept no particles or multiple particles. We demonstrate that in the latter case single particle diffraction patterns can be extracted provided the particles are sufficiently separated. The technique has the potential to greatly increase the amount of data available for three-dimensional imaging of identical particles with X-ray lasers.
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FERMI@Elettra, the first vacuum ultraviolet and soft X-ray free-electron laser (FEL) using by default a "seeded" scheme, became operational in 2011 and has been opened to users since December 2012. The parameters of the seeded FERMI FEL pulses and, in particular, the superior control of emitted radiation in terms of spectral purity and stability meet the stringent requirements for single-shot and resonant coherent diffraction imaging (CDI) experiments. The advantages of the intense seeded FERMI pulses with variable polarization have been demonstrated with the first experiments performed using the multipurpose experimental station operated at the diffraction and projection imaging (DiProI) beamline. The results reported here were obtained with fixed non-periodic targets during the commissioning period in 2012 using 20-32 nm wavelength range. They demonstrate that the performance of the FERMI FEL source and the experimental station meets the requirements of CDI, holography, and resonant magnetic scattering in both multi- and single-shot modes. Moreover, we present the first magnetic scattering experiments employing the fully circularly polarized FERMI pulses. The ongoing developments aim at pushing the lateral resolution by using shorter wavelengths provided by double-stage cascaded FERMI FEL-2 and probing ultrafast dynamic processes using different pump-probe schemes, including jitter-free seed laser pump or FEL-pump∕FEL-probe with two color FEL pulses generated by the same electron bunch.
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Diffractive imaging with free-electron lasers allows structure determination from ensembles of weakly scattering identical nanoparticles. The ultra-short, ultra-bright X-ray pulses provide snapshots of the randomly oriented particles frozen in time, and terminate before the onset of structural damage. As signal strength diminishes for small particles, the synthesis of a three-dimensional diffraction volume requires simultaneous involvement of all data. Here we report the first application of a three-dimensional spatial frequency correlation analysis to carry out this synthesis from noisy single-particle femtosecond X-ray diffraction patterns of nearly identical samples in random and unknown orientations, collected at the Linac Coherent Light Source. Our demonstration uses unsupported test particles created via aerosol self-assembly, and composed of two polystyrene spheres of equal diameter. The correlation analysis avoids the need for orientation determination entirely. This method may be applied to the structural determination of biological macromolecules in solution.
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We successfully use the corners of a common silicon nitride supporting window in lensless X-ray microscopy as extended references in differential holography to obtain a real space hologram of the illuminated object. Moreover, we combine this method with the iterative phasing techniques of coherent diffraction imaging to enhance the spatial resolution on the reconstructed object, and overcome the problem of missing areas in the collected data due to the presence of a beam stop, achieving a resolution close to 85 nm.