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We present time-resolved X-ray absorption spectra of ionized liquid water and demonstrate that OH radicals, H3O+ ions, and solvated electrons all leave distinct X-ray-spectroscopic signatures. Particularly, this allows us to characterize the electron solvation process through a tool that focuses on the electronic response of oxygen atoms in the immediate vicinity of a solvated electron. Our experimental results, supported by ab initio calculations, confirm the formation of a cavity in which the solvated electron is trapped. We show that the solvation dynamics are governed by the magnitude of the random structural fluctuations present in water. As a consequence, the solvation time is highly sensitive to temperature and to the specific way the electron is injected into water.
RESUMEN
We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).
Asunto(s)
Cristalografía/métodos , Complejo de Proteína del Fotosistema II/metabolismo , Ribosomas/metabolismo , Modelos MolecularesRESUMEN
X-ray lasers offer new capabilities in understanding the structure of biological systems, complex materials and matter under extreme conditions. Very short and extremely bright, coherent X-ray pulses can be used to outrun key damage processes and obtain a single diffraction pattern from a large macromolecule, a virus or a cell before the sample explodes and turns into plasma. The continuous diffraction pattern of non-crystalline objects permits oversampling and direct phase retrieval. Here we show that high-quality diffraction data can be obtained with a single X-ray pulse from a non-crystalline biological sample, a single mimivirus particle, which was injected into the pulsed beam of a hard-X-ray free-electron laser, the Linac Coherent Light Source. Calculations indicate that the energy deposited into the virus by the pulse heated the particle to over 100,000 K after the pulse had left the sample. The reconstructed exit wavefront (image) yielded 32-nm full-period resolution in a single exposure and showed no measurable damage. The reconstruction indicates inhomogeneous arrangement of dense material inside the virion. We expect that significantly higher resolutions will be achieved in such experiments with shorter and brighter photon pulses focused to a smaller area. The resolution in such experiments can be further extended for samples available in multiple identical copies.
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Mimiviridae/química , Difracción de Rayos X/instrumentación , Difracción de Rayos X/métodos , Electrones , Calor , Rayos Láser , Fotones , Factores de Tiempo , Rayos XRESUMEN
X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.
Asunto(s)
Cristalografía por Rayos X/métodos , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Unión Proteica , Conformación Proteica/efectos de la radiación , Rayos XRESUMEN
Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.
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Cristalografía por Rayos X/métodos , Cristalografía/métodos , Proteínas/química , Proteínas/ultraestructura , Unión Proteica/efectos de la radiación , Conformación Proteica/efectos de la radiación , Proteínas/efectos de la radiación , Solubilidad/efectos de la radiación , Rayos XRESUMEN
Single shot diffraction imaging experiments via X-ray free-electron lasers can generate as many as hundreds of thousands of diffraction patterns of scattering objects. Recovering the real space contrast of a scattering object from these patterns currently requires a reconstruction process with user guidance in a number of steps, introducing severe bottlenecks in data processing. We present a series of measures that replace user guidance with algorithms that reconstruct contrasts in an unsupervised fashion. We demonstrate the feasibility of automating the reconstruction process by generating hundreds of contrasts obtained from soot particle diffraction experiments.
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Characterizing intense, focused x-ray free electron laser (FEL) pulses is crucial for their use in diffractive imaging. We describe how the distribution of average phase tilts and intensities on hard x-ray pulses with peak intensities of 10(21) W/m(2) can be retrieved from an ensemble of diffraction patterns produced by 70 nm-radius polystyrene spheres, in a manner that mimics wavefront sensors. Besides showing that an adaptive geometric correction may be necessary for diffraction data from randomly injected sample sources, our paper demonstrates the possibility of collecting statistics on structured pulses using only the diffraction patterns they generate and highlights the imperative to study its impact on single-particle diffractive imaging.
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Aerosoles/análisis , Aerosoles/química , Rayos Láser , Fotometría/métodos , Refractometría/métodos , Resonancia por Plasmón de Superficie/métodos , Rayos X , Electrones , Diseño de Equipo , Análisis de Falla de Equipo , MicroesferasRESUMEN
An electrospun liquid microjet has been developed that delivers protein microcrystal suspensions at flow rates of 0.14-3.1 µl min(-1) to perform serial femtosecond crystallography (SFX) studies with X-ray lasers. Thermolysin microcrystals flowed at 0.17 µl min(-1) and diffracted to beyond 4 Å resolution, producing 14,000 indexable diffraction patterns, or four per second, from 140 µg of protein. Nanoflow electrospinning extends SFX to biological samples that necessitate minimal sample consumption.
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Cristalografía por Rayos X/instrumentación , Cristalización , Cristalografía por Rayos X/economía , Campos Electromagnéticos , Diseño de Equipo , Cinética , Rayos Láser , Tamaño de la Muestra , Termolisina/químicaRESUMEN
We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
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Cristalografía por Rayos X/métodos , Ferredoxinas/ultraestructura , Rayos Láser , Nanoestructuras/ultraestructura , Difracción de Rayos X/métodos , Electrones , Conformación Proteica , Rayos XRESUMEN
We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique. Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.
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Single-particle experiments using X-ray Free Electron Lasers produce more than 10(5) snapshots per hour, consisting of an admixture of blank shots (no particle intercepted), and exposures of one or more particles. Experimental data sets also often contain unintentional contamination with different species. We present an unsupervised method able to sort experimental snapshots without recourse to templates, specific noise models, or user-directed learning. The results show 90% agreement with manual classification.
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Transmission-mode direct analysis in real time (TM-DART) is presented as an alternative sampling strategy to traditional methods of sample introduction for DART MS analysis. A custom-designed sample holder was fabricated to rapidly and reproducibly position insecticide-treated nets normal to the ionizing metastable gas stream, enabling transmission of desorbed analyte ions through the holder cavity and into the MS. Introduction of the sample at this fixed geometry eliminates the need for optimizing sample position and allows spectra based on factors such as metastable gas temperature and flow to be systematically evaluated. The results presented here, supported by computational fluid dynamic simulations, demonstrate the effects of these factors on the resulting mass spectra and the potential of this sampling strategy to be used for qualitative and quantitative analyses. Transmission-mode desorption electrospray ionization (TM-DESI) experiments on similar insecticide-treated nets were performed for comparison purposes.
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Mosquiteros Tratados con Insecticida , Malaria/prevención & control , Espectrometría de Masa por Ionización de Electrospray/métodos , Insecticidas/análisis , Insecticidas/química , Nitrilos/análisis , Nitrilos/química , Permetrina/análisis , Permetrina/química , Piretrinas/análisis , Piretrinas/química , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
BACKGROUND: The majority of ovarian cancer biomarker discovery efforts focus on the identification of proteins that can improve the predictive power of presently available diagnostic tests. We here show that metabolomics, the study of metabolic changes in biological systems, can also provide characteristic small molecule fingerprints related to this disease. RESULTS: In this work, new approaches to automatic classification of metabolomic data produced from sera of ovarian cancer patients and benign controls are investigated. The performance of support vector machines (SVM) for the classification of liquid chromatography/time-of-flight mass spectrometry (LC/TOF MS) metabolomic data focusing on recognizing combinations or "panels" of potential metabolic diagnostic biomarkers was evaluated. Utilizing LC/TOF MS, sera from 37 ovarian cancer patients and 35 benign controls were studied. Optimum panels of spectral features observed in positive or/and negative ion mode electrospray (ESI) MS with the ability to distinguish between control and ovarian cancer samples were selected using state-of-the-art feature selection methods such as recursive feature elimination and L1-norm SVM. CONCLUSION: Three evaluation processes (leave-one-out-cross-validation, 12-fold-cross-validation, 52-20-split-validation) were used to examine the SVM models based on the selected panels in terms of their ability for differentiating control vs. disease serum samples. The statistical significance for these feature selection results were comprehensively investigated. Classification of the serum sample test set was over 90% accurate indicating promise that the above approach may lead to the development of an accurate and reliable metabolomic-based approach for detecting ovarian cancer.
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Algoritmos , Cromatografía Liquida/métodos , Biología Computacional/métodos , Espectrometría de Masas/métodos , Metabolómica/métodos , Neoplasias Ováricas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Reconocimiento de Normas Patrones Automatizadas/métodosRESUMEN
BACKGROUND: Counterfeit oral artesunate has been a major public health problem in mainland SE Asia, impeding malaria control. A countrywide stratified random survey was performed to determine the availability and quality of oral artesunate in pharmacies and outlets (shops selling medicines) in the Lao PDR (Laos). METHODS: In 2003, 'mystery' shoppers were asked to buy artesunate tablets from 180 outlets in 12 of the 18 Lao provinces. Outlets were selected using stratified random sampling by investigators not involved in sampling. Samples were analysed for packaging characteristics, by the Fast Red Dye test, high-performance liquid chromatography (HPLC), mass spectrometry (MS), X-ray diffractometry and pollen analysis. RESULTS: Of 180 outlets sampled, 25 (13.9%) sold oral artesunate. Outlets selling artesunate were more commonly found in the more malarious southern Laos. Of the 25 outlets, 22 (88%; 95%CI 68-97%) sold counterfeit artesunate, as defined by packaging and chemistry. No artesunate was detected in the counterfeits by any of the chemical analysis techniques and analysis of the packaging demonstrated seven different counterfeit types. There was complete agreement between the Fast Red dye test, HPLC and MS analysis. A wide variety of wrong active ingredients were found by MS. Of great concern, 4/27 (14.8%) fakes contained detectable amounts of artemisinin (0.26-115.7 mg/tablet). CONCLUSION: This random survey confirms results from previous convenience surveys that counterfeit artesunate is a severe public health problem. The presence of artemisinin in counterfeits may encourage malaria resistance to artemisinin derivatives. With increasing accessibility of artemisinin-derivative combination therapy (ACT) in Laos, the removal of artesunate monotherapy from pharmacies may be an effective intervention.
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Antimaláricos/análisis , Antimaláricos/provisión & distribución , Artemisininas/análisis , Artemisininas/provisión & distribución , Resistencia a Medicamentos , Malaria/tratamiento farmacológico , Malaria/epidemiología , Insuficiencia del Tratamiento , Animales , Antimaláricos/química , Antimaláricos/farmacología , Artemisininas/química , Artemisininas/farmacología , Artesunato , Técnicas de Química Analítica/métodos , Estudios Transversales , Humanos , Laos/epidemiología , Distribución AleatoriaRESUMEN
BACKGROUND: Since 1998 the serious public health problem in South East Asia of counterfeit artesunate, containing no or subtherapeutic amounts of the active antimalarial ingredient, has led to deaths from untreated malaria, reduced confidence in this vital drug, large economic losses for the legitimate manufacturers, and concerns that artemisinin resistance might be engendered. METHODS AND FINDINGS: With evidence of a deteriorating situation, a group of police, criminal analysts, chemists, palynologists, and health workers collaborated to determine the source of these counterfeits under the auspices of the International Criminal Police Organization (INTERPOL) and the Western Pacific World Health Organization Regional Office. A total of 391 samples of genuine and counterfeit artesunate collected in Vietnam (75), Cambodia (48), Lao PDR (115), Myanmar (Burma) (137) and the Thai/Myanmar border (16), were available for analysis. Sixteen different fake hologram types were identified. High-performance liquid chromatography and/or mass spectrometry confirmed that all specimens thought to be counterfeit (195/391, 49.9%) on the basis of packaging contained no or small quantities of artesunate (up to 12 mg per tablet as opposed to approximately 50 mg per genuine tablet). Chemical analysis demonstrated a wide diversity of wrong active ingredients, including banned pharmaceuticals, such as metamizole, and safrole, a carcinogen, and raw material for manufacture of methylenedioxymethamphetamine ('ecstasy'). Evidence from chemical, mineralogical, biological, and packaging analysis suggested that at least some of the counterfeits were manufactured in southeast People's Republic of China. This evidence prompted the Chinese Government to act quickly against the criminal traders with arrests and seizures. CONCLUSIONS: An international multi-disciplinary group obtained evidence that some of the counterfeit artesunate was manufactured in China, and this prompted a criminal investigation. International cross-disciplinary collaborations may be appropriate in the investigation of other serious counterfeit medicine public health problems elsewhere, but strengthening of international collaborations and forensic and drug regulatory authority capacity will be required.
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Artemisia/química , Artemisininas/análisis , Artemisininas/química , Fraude/legislación & jurisprudencia , Internacionalidad/legislación & jurisprudencia , Sesquiterpenos/análisis , Sesquiterpenos/química , Artemisininas/uso terapéutico , Artesunato , Asia Sudoriental/epidemiología , Contaminación de Medicamentos/legislación & jurisprudencia , Contaminación de Medicamentos/prevención & control , Fraude/prevención & control , Humanos , Malaria/tratamiento farmacológico , Malaria/epidemiología , Sesquiterpenos/uso terapéuticoRESUMEN
The internal energy deposition of a Venturi-assisted array of micromachined ultrasonic electrosprays (AMUSE), with and without the application of a DC charging potential, is compared with equivalent experiments for Venturi-assisted electrospray ionization (ESI) using the "survival yield" method on a series of para-substituted benzylpyridinium salts. Under conditions previously shown to provide maximum ion yields for standard compounds, the observed mean internal energies were nearly identical (1.93-2.01 eV). Operation of AMUSE without nitrogen flow to sustain the air amplifier focusing effect generated energetically colder ions with mean internal energies that were up to 39% lower than those for ESI. A balance between improved ion transfer, adequate desolvation, and favorable ion energetics was achieved by selection of optimum operational ranges for the parameters that most strongly influence the ion population: the air amplifier gas flow rate and API capillary temperature. Examination of the energy landscapes obtained for combinations of these parameters showed that a low internal energy region (Asunto(s)
Espectrometría de Masa por Ionización de Electrospray/instrumentación
, Espectrometría de Masa por Ionización de Electrospray/métodos
, Diseño de Equipo
, Compuestos de Piridinio/química
, Termodinámica
, Ultrasonido
RESUMEN
Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
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Mimiviridae , Difracción de Rayos X , Algoritmos , Simulación por Computador , Cristalografía por Rayos X , Recolección de Datos , Electrones , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Rayos Láser , Modelos Teóricos , Tamaño de la Partícula , Dispersión de Radiación , Rayos XRESUMEN
We present an analysis of ice nucleation kinetics from near-ambient pressure water as temperature decreases below the homogeneous limit TH by cooling micrometer-sized droplets (microdroplets) evaporatively at 103-104 K/s and probing the structure ultrafast using femtosecond pulses from the Linac Coherent Light Source (LCLS) free-electron X-ray laser. Below 232 K, we observed a slower nucleation rate increase with decreasing temperature than anticipated from previous measurements, which we suggest is due to the rapid decrease in water's diffusivity. This is consistent with earlier findings that microdroplets do not crystallize at <227 K, but vitrify at cooling rates of 106-107 K/s. We also hypothesize that the slower increase in the nucleation rate is connected with the proposed "fragile-to-strong" transition anomaly in water.
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X-ray free-electron lasers have enabled new approaches to the structural determination of protein crystals that are too small or radiation-sensitive for conventional analysis1. For sufficiently short pulses, diffraction is collected before significant changes occur to the sample, and it has been predicted that pulses as short as 10 fs may be required to acquire atomic-resolution structural information1-4. Here, we describe a mechanism unique to ultrafast, ultra-intense X-ray experiments that allows structural information to be collected from crystalline samples using high radiation doses without the requirement for the pulse to terminate before the onset of sample damage. Instead, the diffracted X-rays are gated by a rapid loss of crystalline periodicity, producing apparent pulse lengths significantly shorter than the duration of the incident pulse. The shortest apparent pulse lengths occur at the highest resolution, and our measurements indicate that current X-ray free-electron laser technology5 should enable structural determination from submicrometre protein crystals with atomic resolution.
RESUMEN
Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.