Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

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.

Ultrafast X-ray probing of water structure below the homogeneous ice nucleation temperature.

Water has a number of anomalous physical properties, and some of these become drastically enhanced on supercooling below the freezing point. Particular interest has focused on thermodynamic response functions that can be described using a normal component and an anomalous component that seems to diverge at about 228 kelvin (refs 1-3). This has prompted debate about conflicting theories that aim to explain many of the anomalous thermodynamic properties of water. One popular theory attributes the divergence to a phase transition between two forms of liquid water occurring in the 'no man's land' that lies below the homogeneous ice nucleation temperature (TH) at approximately 232 kelvin and above about 160 kelvin, and where rapid ice crystallization has prevented any measurements of the bulk liquid phase. In fact, the reliable determination of the structure of liquid water typically requires temperatures above about 250 kelvin. Water crystallization has been inhibited by using nanoconfinement, nanodroplets and association with biomolecules to give liquid samples at temperatures below TH, but such measurements rely on nanoscopic volumes of water where the interaction with the confining surfaces makes the relevance to bulk water unclear. Here we demonstrate that femtosecond X-ray laser pulses can be used to probe the structure of liquid water in micrometre-sized droplets that have been evaporatively cooled below TH. We find experimental evidence for the existence of metastable bulk liquid water down to temperatures of 227(-1)(+2) kelvin in the previously largely unexplored no man's land. We observe a continuous and accelerating increase in structural ordering on supercooling to approximately 229 kelvin, where the number of droplets containing ice crystals increases rapidly. But a few droplets remain liquid for about a millisecond even at this temperature. The hope now is that these observations and our detailed structural data will help identify those theories that best describe and explain the behaviour of water.

Fractal morphology, imaging and mass spectrometry of single aerosol particles in flight.

The morphology of micrometre-size particulate matter is of critical importance in fields ranging from toxicology to climate science, yet these properties are surprisingly difficult to measure in the particles' native environment. Electron microscopy requires collection of particles on a substrate; visible light scattering provides insufficient resolution; and X-ray synchrotron studies have been limited to ensembles of particles. Here we demonstrate an in situ method for imaging individual sub-micrometre particles to nanometre resolution in their native environment, using intense, coherent X-ray pulses from the Linac Coherent Light Source free-electron laser. We introduced individual aerosol particles into the pulsed X-ray beam, which is sufficiently intense that diffraction from individual particles can be measured for morphological analysis. At the same time, ion fragments ejected from the beam were analysed using mass spectrometry, to determine the composition of single aerosol particles. Our results show the extent of internal dilation symmetry of individual soot particles subject to non-equilibrium aggregation, and the surprisingly large variability in their fractal dimensions. More broadly, our methods can be extended to resolve both static and dynamic morphology of general ensembles of disordered particles. Such general morphology has implications in topics such as solvent accessibilities in proteins, vibrational energy transfer by the hydrodynamic interaction of amino acids, and large-scale production of nanoscale structures by flame synthesis.

Femtosecond dark-field imaging with an X-ray free electron laser.

The emergence of femtosecond diffractive imaging with X-ray lasers has enabled pioneering structural studies of isolated particles, such as viruses, at nanometer length scales. However, the issue of missing low frequency data significantly limits the potential of X-ray lasers to reveal sub-nanometer details of micrometer-sized samples. We have developed a new technique of dark-field coherent diffractive imaging to simultaneously overcome the missing data issue and enable us to harness the unique contrast mechanisms available in dark-field microscopy. Images of airborne particulate matter (soot) up to two microns in length were obtained using single-shot diffraction patterns obtained at the Linac Coherent Light Source, four times the size of objects previously imaged in similar experiments. This technique opens the door to femtosecond diffractive imaging of a wide range of micrometer-sized materials that exhibit irreproducible complexity down to the nanoscale, including airborne particulate matter, small cells, bacteria and gold-labeled biological samples.

In vitro study of antimicrobial activity of some plant seeds against bacterial strains causing food poisoning diseases.

In this research, some plant seeds powder was evaluated to find their potential effect to rule diseases of food poisoning. Antimicrobial effect of five plant seeds was examined contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella. pneumonia and Candida albicans by using well diffusion method. Antimicrobial activity studies revealed high potential activity of plant seeds powder of Nigella sativa L., cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum with variable efficiency contra tested microbial strains with concentration of 100 mg/ml, except Sesamum radiatum scored no effect. The T. foenum and N. sativa seed powder showed the largest inhibition zone (24-20 mm) contra K. pneumonia, followed by S. aureus (20-18 mm) and C. albicans (15mm) respectively. The five plant seeds powder exhibited bacteriostatic and bactericidal effects with MIC's 20 and MBC 40 mg/ml against K. pneumonia, and MIC's 40 and MBC 60 mg/ml against S. aureus. The results of this study indicated that plants seeds powder have promising antimicrobial activities and their potential applications in food process. It could be utilized as a natural medicinal alternative instead of chemical substance.

Cryptotomography: reconstructing 3D Fourier intensities from randomly oriented single-shot diffraction patterns.

We reconstructed the 3D Fourier intensity distribution of monodisperse prolate nanoparticles using single-shot 2D coherent diffraction patterns collected at DESY's FLASH facility when a bright, coherent, ultrafast x-ray pulse intercepted individual particles of random, unmeasured orientations. This first experimental demonstration of cryptotomography extended the expansion-maximization-compression framework to accommodate unmeasured fluctuations in photon fluence and loss of data due to saturation or background scatter. This work is an important step towards realizing single-shot diffraction imaging of single biomolecules.

In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

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.

In vitro study of antimicrobial activity of some plant seeds against bacterial strains causing food poisoning diseases / Estudo in vitro da atividade antimicrobiana de algumas sementes de plantas contra cepas bacterianas que causam doenças de intoxicação alimentar

In this research, some plant seeds powder was evaluated to find their potential effect to rule diseases of food poisoning. Antimicrobial effect of five plant seeds was examined contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella. pneumonia and Candida albicans by using well diffusion method. Antimicrobial activity studies revealed high potential activity of plant seeds powder of Nigella sativa L., cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum with variable efficiency contra tested microbial strains with concentration of 100 mg/ml, except Sesamum radiatum scored no effect. The T. foenum and N. sativa seed powder showed the largest inhibition zone (24-20 mm) contra K. pneumonia, followed by S. aureus (20-18 mm) and C. albicans (15mm) respectively. The five plant seeds powder exhibited bacteriostatic and bactericidal effects with MIC's 20 and MBC 40 mg/ml against K. pneumonia, and MIC's 40 and MBC 60 mg/ml against S. aureus. The results of this study indicated that plants seeds powder have promising antimicrobial activities and their potential applications in food process. It could be utilized as a natural medicinal alternative instead of chemical substance.

Nesta pesquisa, o pó de sementes de plantas foi avaliado para encontrar seu efeito potencial no controle de doenças de intoxicação alimentar. O efeito antimicrobiano de cinco sementes de plantas foi examinado contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia e Candida albicans usando o método de difusão bem. Estudos de atividade antimicrobiana revelaram alto potencial de atividade de sementes de plantas em pó de Nigella sativa L., Cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum com eficiência variável contra cepas microbianas testadas com concentração de 100 mg / ml, exceto Sesamum radiatum com pontuação não efeito. O pó de sementes de T. foenum e N. sativa apresentou a maior zona de inibição (24-20 mm) contra K. pneumonia, seguido por S. aureus (20-18 mm) e C. albicans (15 mm), respectivamente. O pó de cinco sementes de plantas exibiu efeitos bacteriostáticos e bactericidas com MIC's 20 e MBC 40 mg / ml contra K. pneumonia, enquanto MIC's 40 e MBC 60 mg / ml contra S. aureus. Os resultados deste estudo indicaram que os pós de sementes de plantas apresentam promissoras atividades antimicrobianas e suas potenciais aplicações em processos alimentícios. Ele poderia ser utilizado como alternativa medicinal natural em vez de substância química.

In vitro study of antimicrobial activity of some plant seeds against bacterial strains causing food poisoning diseases

Abstract In this research, some plant seeds powder was evaluated to find their potential effect to rule diseases of food poisoning. Antimicrobial effect of five plant seeds was examined contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella. pneumonia and Candida albicans by using well diffusion method. Antimicrobial activity studies revealed high potential activity of plant seeds powder of Nigella sativa L., cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum with variable efficiency contra tested microbial strains with concentration of 100 mg/ml, except Sesamum radiatum scored no effect. The T. foenum and N. sativa seed powder showed the largest inhibition zone (24-20 mm) contra K. pneumonia, followed by S. aureus (20-18 mm) and C. albicans (15mm) respectively. The five plant seeds powder exhibited bacteriostatic and bactericidal effects with MICs 20 and MBC 40 mg/ml against K. pneumonia, and MICs 40 and MBC 60 mg/ml against S. aureus. The results of this study indicated that plants seeds powder have promising antimicrobial activities and their potential applications in food process. It could be utilized as a natural medicinal alternative instead of chemical substance.

Resumo Nesta pesquisa, o pó de sementes de plantas foi avaliado para encontrar seu efeito potencial no controle de doenças de intoxicação alimentar. O efeito antimicrobiano de cinco sementes de plantas foi examinado contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia e Candida albicans usando o método de difusão bem. Estudos de atividade antimicrobiana revelaram alto potencial de atividade de sementes de plantas em pó de Nigella sativa L., Cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum com eficiência variável contra cepas microbianas testadas com concentração de 100 mg / ml, exceto Sesamum radiatum com pontuação não efeito. O pó de sementes de T. foenum e N. sativa apresentou a maior zona de inibição (24-20 mm) contra K. pneumonia, seguido por S. aureus (20-18 mm) e C. albicans (15 mm), respectivamente. O pó de cinco sementes de plantas exibiu efeitos bacteriostáticos e bactericidas com MICs 20 e MBC 40 mg / ml contra K. pneumonia, enquanto MICs 40 e MBC 60 mg / ml contra S. aureus. Os resultados deste estudo indicaram que os pós de sementes de plantas apresentam promissoras atividades antimicrobianas e suas potenciais aplicações em processos alimentícios. Ele poderia ser utilizado como alternativa medicinal natural em vez de substância química.

In vitro study of antimicrobial activity of some plant seeds against bacterial strains causing food poisoning diseases / Estudo in vitro da atividade antimicrobiana de algumas sementes de plantas contra cepas bacterianas que causam doenças de intoxicação alimentar

In this research, some plant seeds powder was evaluated to find their potential effect to rule diseases of food poisoning. Antimicrobial effect of five plant seeds was examined contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella. pneumonia and Candida albicans by using well diffusion method. Antimicrobial activity studies revealed high potential activity of plant seeds powder of Nigella sativa L., cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum with variable efficiency contra tested microbial strains with concentration of 100 mg/ml, except Sesamum radiatum scored no effect. The T. foenum and N. sativa seed powder showed the largest inhibition zone (24-20 mm) contra K. pneumonia, followed by S. aureus (20-18 mm) and C. albicans (15mm) respectively. The five plant seeds powder exhibited bacteriostatic and bactericidal effects with MIC's 20 and MBC 40 mg/ml against K. pneumonia, and MIC's 40 and MBC 60 mg/ml against S. aureus. The results of this study indicated that plants seeds powder have promising antimicrobial activities and their potential applications in food process. It could be utilized as a natural medicinal alternative instead of chemical substance.

Nesta pesquisa, o pó de sementes de plantas foi avaliado para encontrar seu efeito potencial no controle de doenças de intoxicação alimentar. O efeito antimicrobiano de cinco sementes de plantas foi examinado contra Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia e Candida albicans usando o método de difusão bem. Estudos de atividade antimicrobiana revelaram alto potencial de atividade de sementes de plantas em pó de Nigella sativa L., Cucurbita pepo, Sesamum radiatum, Trigonella foenum-graecum, Linum usitatissimum com eficiência variável contra cepas microbianas testadas com concentração de 100 mg / ml, exceto Sesamum radiatum com pontuação não efeito. O pó de sementes de T. foenum e N. sativa apresentou a maior zona de inibição (24-20 mm) contra K. pneumonia, seguido por S. aureus (20-18 mm) e C. albicans (15 mm), respectivamente. O pó de cinco sementes de plantas exibiu efeitos bacteriostáticos e bactericidas com MIC's 20 e MBC 40 mg / ml contra K. pneumonia, enquanto MIC's 40 e MBC 60 mg / ml contra S. aureus. Os resultados deste estudo indicaram que os pós de sementes de plantas apresentam promissoras atividades antimicrobianas e suas potenciais aplicações em processos alimentícios. Ele poderia ser utilizado como alternativa medicinal natural em vez de substância química.

Electronic damage in S atoms in a native protein crystal induced by an intense X-ray free-electron laser pulse.

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.

The linac coherent light source single particle imaging road map.

Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.

Toward atomic resolution diffractive imaging of isolated molecules with X-ray free-electron lasers.

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.

Single-particle structure determination by correlations of snapshot X-ray diffraction patterns.

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.

Three-dimensional coherent x-ray diffraction imaging of a ceramic nanofoam: determination of structural deformation mechanisms.

Ultralow density polymers, metals, and ceramic nanofoams are valued for their high strength-to-weight ratio, high surface area, and insulating properties ascribed to their structural geometry. We obtain the labrynthine internal structure of a tantalum oxide nanofoam by x-ray diffractive imaging. Finite-element analysis from the structure reveals mechanical properties consistent with bulk samples and with a diffusion-limited cluster aggregation model, while excess mass on the nodes discounts the dangling fragments hypothesis of percolation theory.

Damped and thermal motion of laser-aligned hydrated macromolecule beams for diffraction.

We consider a monodispersed Rayleigh droplet beam of water droplets doped with proteins. An intense infrared laser is used to align these droplets. The arrangement has been proposed for electron- and x-ray-diffraction studies of proteins which are difficult to crystallize. This paper considers the effect of thermal fluctuations on the angular spread of alignment in thermal equilibrium, and relaxation phenomena, particularly the damping of oscillations excited as the molecules enter the field. The possibility of adiabatic alignment is also considered. We find that damping times in a high-pressure gas cell as used in x-ray-diffraction experiments are short compared with the time taken for molecules to traverse the beam and that a suitably shaped field might be used for electron-diffraction experiments in vacuum to provide adiabatic alignment, thus obviating the need for a damping gas cell.

Quantitative optical phase microscopy.

We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.

Quantitative phase-amplitude microscopy I: optical microscopy.

In this paper, the application of a new optical microscopy method (quantitative phase-amplitude microscopy) to biological imaging is explored, and the issue of resolution and image quality is examined. The paper begins by presenting a theoretical analysis of the method using the optical transfer function formalism of Streibl (1985). The effect of coherence on the formation of the phase image is explored, and it is shown that the resolution of the method is not compromised over that of a conventional bright-field image. It is shown that the signal-to-noise ratio of the phase recovery, however, does depend on the degree of coherence in the illumination. Streibl (1985) notes that partially coherent image formation is a non-linear process because of the intermingling of amplitude and phase information. The work presented here shows that the quantitative phase-amplitude microscopy method acts to linearize the image formation process, and that the phase and amplitude information is properly described using a transfer function analysis. The theoretical conclusions are tested experimentally using an optical microscope and the theoretical deductions are confirmed. Samples for microscopy influence both the phase and amplitude of the light wave and it is demonstrated that the new phase recovery method can separate the amplitude and phase information, something not possible using traditional phase microscopy. In the case of a coherent wave, knowledge of the phase and amplitude constitutes complete information that can be used to emulate other forms of microscopy. This capacity is demonstrated by recovering the phase of a sample and using the data to emulate a differential interference contrast image.

Quantitative phase-amplitude microscopy. III. The effects of noise.

We explore the effect of noise on images obtained using quantitative phase-amplitude microscopy - a new microscopy technique based on the determination of phase from the intensity evolution of propagating radiation. We compare the predictions with experimental results and also propose an approach that allows good-quality quantitative phase retrieval to be obtained even for very noisy data.

Noninterferometric quantitative phase imaging with soft x rays.

We demonstrate quantitative noninterferometric x-ray phase-amplitude measurement. We present results from two experimental geometries. The first geometry uses x rays diverging from a point source to produce high-resolution holograms of submicrometer-sized objects. The measured phase of the projected image agrees with the geometrically determined phase to within +/-7%. The second geometry uses a direct imaging microscope setup that allows the formation of a magnified image with a zone-plate lens. Here a direct measure of the object phase is made and agrees with that of the magnified object to better than +/-10%. In both cases the accuracy of the phase is limited by the pixel resolution.