RESUMEN
Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Receptor de Angiotensina Tipo 1/química , Secuencia de Aminoácidos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutagénesis , Naftiridinas/química , Naftiridinas/farmacología , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Alineación de SecuenciaRESUMEN
Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Eosinófilos/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Celulitis (Flemón)/metabolismo , Celulitis (Flemón)/patología , Proteínas de Unión al ADN/toxicidad , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Eosinofilia/metabolismo , Eosinofilia/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Escherichia coli/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/fisiología , Ratones Endogámicos C57BL , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Vesículas Secretoras/metabolismo , Piel/efectos de los fármacos , Piel/patologíaRESUMEN
BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.
Asunto(s)
Bacillus/química , Toxinas Bacterianas/química , Culicidae , Insecticidas/química , Larva , Rayos Láser , Animales , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Culicidae/metabolismo , Concentración de Iones de Hidrógeno , Larva/química , Larva/metabolismo , Modelos Moleculares , Multimerización de Proteína , Proteolisis , Tirosina/químicaRESUMEN
The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face ß-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.
Asunto(s)
Nanopartículas/química , Nanopartículas/toxicidad , alfa-Sinucleína/química , alfa-Sinucleína/toxicidad , Amiloide/química , Microscopía por Crioelectrón , Electrones , Humanos , Cuerpos de Lewy/química , Modelos Moleculares , Enfermedad de Parkinson , Estructura Terciaria de Proteína , Dispersión de RadiaciónRESUMEN
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
Asunto(s)
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Humanos , Rayos Láser , Ratones , Modelos Moleculares , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Transducción de Señal , Rayos XRESUMEN
The bright ultrafast pulses of X-ray Free-Electron Lasers allow investigation into the structure of matter under extreme conditions. We have used single pulses to ionize and probe water as it undergoes a phase transition from liquid to plasma. We report changes in the structure of liquid water on a femtosecond time scale when irradiated by single 6.86 keV X-ray pulses of more than 106 J/cm2 These observations are supported by simulations based on molecular dynamics and plasma dynamics of a water system that is rapidly ionized and driven out of equilibrium. This exotic ionic and disordered state with the density of a liquid is suggested to be structurally different from a neutral thermally disordered state.
Asunto(s)
Electrones , Calor , Rayos Láser , Agua/química , Cristalografía , Simulación de Dinámica Molecular , Factores de TiempoRESUMEN
The determination of protein crystal structures is hampered by the need for macroscopic crystals. X-ray free-electron lasers (FELs) provide extremely intense pulses of femtosecond duration, which allow data collection from nanometre- to micrometre-sized crystals in a 'diffraction-before-destruction' approach. So far, all protein structure determinations carried out using FELs have been based on previous knowledge of related, known structures. Here we show that X-ray FEL data can be used for de novo protein structure determination, that is, without previous knowledge about the structure. Using the emerging technique of serial femtosecond crystallography, we performed single-wavelength anomalous scattering measurements on microcrystals of the well-established model system lysozyme, in complex with a lanthanide compound. Using Monte-Carlo integration, we obtained high-quality diffraction intensities from which experimental phases could be determined, resulting in an experimental electron density map good enough for automated building of the protein structure. This demonstrates the feasibility of determining novel protein structures using FELs. We anticipate that serial femtosecond crystallography will become an important tool for the structure determination of proteins that are difficult to crystallize, such as membrane proteins.
Asunto(s)
Cristalografía/métodos , Electrones , Rayos Láser , Proteínas/química , Difracción de Rayos X/métodos , Rayos X , Animales , Pollos , Cristalización , Femenino , Gadolinio , Proteínas de la Membrana/química , Modelos Moleculares , Método de Montecarlo , Muramidasa/química , Conformación Proteica , Factores de TiempoRESUMEN
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
Asunto(s)
Cristalografía por Rayos X , Cianobacterias/química , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , Estructura Terciaria de ProteínaRESUMEN
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Receptor de Serotonina 5-HT2B/química , Receptor de Serotonina 5-HT2B/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Dominio Catalítico , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Ergotamina/química , Ergotamina/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Conformación Proteica , Receptor de Serotonina 5-HT2B/metabolismo , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/metabolismoRESUMEN
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
Asunto(s)
Cristalografía/métodos , Electrones , Granulovirus/ultraestructura , Péptidos y Proteínas de Señalización Intercelular/química , Rayos Láser , Cristalografía/instrumentación , Granulovirus/química , Modelos Moleculares , Progranulinas , Estructura Secundaria de Proteína , SincrotronesRESUMEN
The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.
RESUMEN
There is a fundamental interest in studying photoinduced dynamics in nanoparticles and nanostructures as it provides insight into their mechanical and thermal properties out of equilibrium and during phase transitions. Nanoparticles can display significantly different properties from the bulk, which is due to the interplay between their size, morphology, crystallinity, defect concentration, and surface properties. Particularly interesting scenarios arise when nanoparticles undergo phase transitions, such as melting induced by an optical laser. Current theoretical evidence suggests that nanoparticles can undergo reversible nonhomogenous melting with the formation of a core-shell structure consisting of a liquid outer layer. To date, studies from ensembles of nanoparticles have tentatively suggested that such mechanisms are present. Here we demonstrate imaging transient melting and softening of the acoustic phonon modes of an individual gold nanocrystal, using an X-ray free electron laser. The results demonstrate that the transient melting is reversible and nonhomogenous, consistent with a core-shell model of melting. The results have implications for understanding transient processes in nanoparticles and determining their elastic properties as they undergo phase transitions.
RESUMEN
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
Asunto(s)
Transferencia de Energía/efectos de la radiación , Rayos Láser , Ficobiliproteínas/efectos de la radiación , Ficobiliproteínas/ultraestructura , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Ficobiliproteínas/química , Conformación Proteica/efectos de la radiación , Dosis de RadiaciónRESUMEN
X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.
Asunto(s)
Rayos Láser , Sustancias Macromoleculares/química , Bacillus/enzimología , Calcio/química , Calibración , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Electrones , Diseño de Equipo , Funciones de Verosimilitud , Modelos Químicos , Conformación Molecular , Muramidasa/química , Nanotecnología , Reproducibilidad de los Resultados , Programas Informáticos , Termolisina/química , Rayos X , Zinc/químicaRESUMEN
Determining fluctuations in focus properties is essential for many experiments at Self-Amplified-Spontaneous-Emission (SASE) based Free-Electron-Lasers (FELs), in particular for imaging single non-crystalline biological particles. We report on a diffractive imaging technique to fully characterize highly focused, single-shot pulses using an iterative phase retrieval algorithm, and benchmark it against an existing Hartmann wavefront sensor. The results, both theoretical and experimental, demonstrate the effectiveness of this technique to provide a comprehensive and convenient shot-to-shot measurement of focused-pulse wave fields and source-point positional variations without the need for manipulative optics between the focus and the detector.
RESUMEN
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (â¼200 nm to 2 µm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
Asunto(s)
Cristalografía por Rayos X/métodos , Nanopartículas/química , Nanotecnología/métodos , Complejo de Proteína del Fotosistema I/química , Cristalografía por Rayos X/instrumentación , Rayos Láser , Modelos Moleculares , Nanotecnología/instrumentación , Conformación Proteica , Factores de Tiempo , Rayos XRESUMEN
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.
Asunto(s)
Mimiviridae/química , Difracción de Rayos X/instrumentación , Difracción de Rayos X/métodos , Electrones , Calor , Rayos Láser , Fotones , Factores de Tiempo , Rayos XRESUMEN
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (â¼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Endotoxinas/química , Proteínas Hemolisinas/química , Esporas Bacterianas/química , Bacillus thuringiensis/ultraestructura , Toxinas de Bacillus thuringiensis , Cristalización , Cristalografía por Rayos X/instrumentación , Rayos Láser , Esporas Bacterianas/ultraestructura , Sincrotrones , Difracción de 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.
Asunto(s)
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
X-ray free-electron laser sources such as the Linac Coherent Light Source offer very exciting possibilities for unique research. However, beam time at such facilities is very limited and in high demand. This has led to significant efforts towards beam multiplexing of various forms. One such effort involves re-using the so-called spent beam that passes through the hole in an area detector after a weak interaction with a primary sample. This beam can be refocused into a secondary interaction region and used for a second, independent experiment operating in series. The beam profile of this refocused beam was characterized for a particular experimental geometry at the Coherent X-ray Imaging instrument at LCLS. A demonstration of this multiplexing capability was performed with two simultaneous serial femtosecond crystallography experiments, both yielding interpretable data of sufficient quality to produce electron density maps.