RESUMEN
Directional transport of protons across an energy transducing membrane-proton pumping-is ubiquitous in biology. Bacteriorhodopsin (bR) is a light-driven proton pump that is activated by a buried all-trans retinal chromophore being photoisomerized to a 13-cis conformation. The mechanism by which photoisomerization initiates directional proton transport against a proton concentration gradient has been studied by a myriad of biochemical, biophysical, and structural techniques. X-ray free electron lasers (XFELs) have created new opportunities to probe the structural dynamics of bR at room temperature on timescales from femtoseconds to milliseconds using time-resolved serial femtosecond crystallography (TR-SFX). Wereview these recent developments and highlight where XFEL studies reveal new details concerning the structural mechanism of retinal photoisomerization and proton pumping. We also discuss the extent to which these insights were anticipated by earlier intermediate trapping studies using synchrotron radiation. TR-SFX will open up the field for dynamical studies of other proteins that are not naturally light-sensitive.
Asunto(s)
Bacteriorodopsinas/ultraestructura , Rayos Láser , Protones , Retinaldehído/química , Difracción de Rayos X/métodos , Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Cristalografía/instrumentación , Cristalografía/métodos , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Transporte Iónico , Modelos Moleculares , Conformación Proteica , Retinaldehído/metabolismo , Sincrotrones/instrumentación , Rayos XRESUMEN
Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.
Asunto(s)
Canales de Cloruro/química , Rayos Láser , Canales de Cloruro/metabolismo , Cristalografía , Citoplasma/metabolismo , Transporte Iónico , Luz , Conformación Proteica , Rayos XRESUMEN
The advent of X-ray Free Electron Lasers (XFELs) has ushered in a transformative era in the field of structural biology, materials science, and ultrafast physics. These state-of-the-art facilities generate ultra-bright, femtosecond-long X-ray pulses, allowing researchers to delve into the structure and dynamics of molecular systems with unprecedented temporal and spatial resolutions. The unique properties of XFEL pulses have opened new avenues for scientific exploration that were previously considered unattainable. One of the most notable applications of XFELs is in structural biology. Traditional X-ray crystallography, while instrumental in determining the structures of countless biomolecules, often requires large, high-quality crystals and may not capture highly transient states of proteins. XFELs, with their ability to produce diffraction patterns from nanocrystals or even single particles, have provided solutions to these challenges. XFEL has expanded the toolbox of structural biologists by enabling structural determination approaches such as Single Particle Imaging (SPI) and Serial X-ray Crystallography (SFX). Despite their remarkable capabilities, the journey of XFELs is still in its nascent stages, with ongoing advancements aimed at improving their coherence, pulse duration, and wavelength tunability.
Asunto(s)
Electrones , Proteínas , Cristalografía por Rayos X , Proteínas/química , Rayos X , Rayos LáserRESUMEN
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Rodopsinas Microbianas/metabolismo , Cationes Monovalentes/metabolismo , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/efectos de la radiación , Canales de Cloruro/ultraestructura , Cristalografía/métodos , Radiación Electromagnética , Rayos Láser , Simulación de Dinámica Molecular , Nocardioides , Conformación Proteica en Hélice alfa/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Proteínas Recombinantes/ultraestructura , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsinas Microbianas/aislamiento & purificación , Rodopsinas Microbianas/efectos de la radiación , Rodopsinas Microbianas/ultraestructura , Agua/metabolismoRESUMEN
Serial Femtosecond Crystallography at the X-ray Free Electron Laser (XFEL) sources enabled the imaging of the catalytic intermediates of the oxygen evolution reaction of Photosystem II (PSII). However, due to the incoherent transition of the S-states, the resolved structures are a convolution from different catalytic states. Here, we train Decision Tree Classifier and K-means clustering models on Mn compounds obtained from the Cambridge Crystallographic Database to predict the S-state of the X-ray, XFEL, and CryoEM structures by predicting the Mn's oxidation states in the oxygen-evolving complex. The model agrees mostly with the XFEL structures in the dark S1 state. However, significant discrepancies are observed for the excited XFEL states (S2, S3, and S0) and the dark states of the X-ray and CryoEM structures. Furthermore, there is a mismatch between the predicted S-states within the two monomers of the same dimer, mainly in the excited states. We validated our model against other metalloenzymes, the valence bond model and the Mn spin densities calculated using density functional theory for two of the mismatched predictions of PSII. The model suggests designing a more optimized sample delivery and illumiation systems are crucial to precisely resolve the geometry of the advanced S-states to overcome the noncoherent S-state transition. In addition, significant radiation damage is observed in X-ray and CryoEM structures, particularly at the dangler Mn center (Mn4). Our model represents a valuable tool for investigating the electronic structure of the catalytic metal cluster of PSII to understand the water splitting mechanism.
Asunto(s)
Complejo de Proteína del Fotosistema II , Aprendizaje Automático no Supervisado , Complejo de Proteína del Fotosistema II/metabolismo , Oxidación-Reducción , Iones , Oxígeno/química , Agua/química , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Ferritin-like proteins share a common fold, a four α-helix bundle core, often coordinating a pair of metal ions. Although conserved, the ferritin fold permits a diverse set of reactions, and is central in a multitude of macromolecular enzyme complexes. Here, we emphasize this diversity through three members of the ferritin-like superfamily: the soluble methane monooxygenase, the class I ribonucleotide reductase and the aldehyde deformylating oxygenase. They all rely on dinuclear metal cofactors to catalyze different challenging oxygen-dependent reactions through the formation of multi-protein complexes. Recent studies using cryo-electron microscopy, serial femtosecond crystallography at an X-ray free electron laser source, or single-crystal X-ray diffraction, have reported the structures of the active protein complexes, and revealed unprecedented insights into the molecular mechanisms of these three enzymes.
Asunto(s)
Ferritinas , Ribonucleótido Reductasas , Aldehídos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Ferritinas/metabolismo , Iones/metabolismo , Complejos Multienzimáticos/metabolismo , Oxígeno/metabolismo , Oxigenasas/química , Oxigenasas/metabolismo , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismoRESUMEN
Currently, X-ray crystallography, which typically uses synchrotron sources, remains the dominant method for structural determination of proteins and other biomolecules. However, small protein crystals do not provide sufficiently high-resolution diffraction patterns and suffer radiation damage; therefore, conventional X-ray crystallography needs larger protein crystals. The burgeoning method of serial crystallography using X-ray free-electron lasers (XFELs) avoids these challenges: it affords excellent structural data from weakly diffracting objects, including tiny crystals. An XFEL is implemented by irradiating microjets of suspensions of microcrystals with very intense X-ray beams. However, while the method for creating microcrystalline microjets is well established, little attention is given to the growth of high-quality nano/microcrystals suitable for XFEL experiments. In this study, in order to assist the growth of such crystals, we calculate the mean crystal size and the time needed to grow crystals to the desired size in batch crystallization (the predominant method for preparing the required microcrystalline slurries); this time is reckoned theoretically both for microcrystals and for crystals larger than the upper limit of the Gibbs-Thomson effect. The impact of the omnipresent impurities on the growth of microcrystals is also considered quantitatively. Experiments, performed with the model protein lysozyme, support the theoretical predictions.
Asunto(s)
Electrones , Sincrotrones , Rayos X , Cristalografía por Rayos X , Proteínas , Rayos LáserRESUMEN
Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined inâ vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both inâ vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.
Asunto(s)
Escherichia coli , Microscopía , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/químicaRESUMEN
Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding.
Asunto(s)
Nucleótidos de Desoxiguanina/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , GTP Fosfohidrolasas/química , Sitio Alostérico , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Nucleótidos de Desoxiguanina/química , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Modelos Moleculares , Proteína 1 que Contiene Dominios SAM y HD/química , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Especificidad por SustratoRESUMEN
Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Manejo de Especímenes/métodos , Alcohol Deshidrogenasa/química , Cristalización , Endo-1,4-beta Xilanasas/química , Endopeptidasa K/química , Proteínas de Plantas/química , Conformación ProteicaRESUMEN
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
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
Asunto(s)
Actinomycetales/química , Canales de Cloruro/química , Rodopsinas Microbianas/química , Cristalografía por Rayos X , Dominios ProteicosRESUMEN
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
Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of â¼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by â¼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Animales , Monóxido de Carbono/metabolismo , Bovinos , Cristalografía por Rayos X , Complejo IV de Transporte de Electrones/química , Conformación ProteicaRESUMEN
Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compoundâ I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.
Asunto(s)
Arginina/metabolismo , Colorantes/metabolismo , Hemo/metabolismo , Compuestos de Hierro/metabolismo , Oxígeno/metabolismo , Peroxidasa/metabolismo , Arginina/química , Biocatálisis , Colorantes/química , Cristalografía por Rayos X , Hemo/química , Compuestos de Hierro/química , Modelos Moleculares , Oxidación-Reducción , Oxígeno/química , Peroxidasa/química , Streptomyces lividans/enzimologíaRESUMEN
The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.
RESUMEN
Ultrafast pump-probe X-ray crystallography has now been established at X-ray free electron lasers that operate at hard X-ray energies. We discuss the performance and development of current applications in terms of the available data quality and sensitivity to detect and analyse structural dynamics. A discussion of technical capabilities expected at future high repetition rate applications as well as future non-collinear multi-pulse schemes focuses on the possibility to advance the technique to the practical application of the X-ray crystallographic equivalent of an impulse time-domain Raman measurement of vibrational coherence. Furthermore, we present calculations of the magnitude of population differences and distributions prepared with ultrafast optical pumping of single crystals in the typical serial femtosecond crystallography geometry, which are developed for the general uniaxial and biaxial cases. The results present opportunities for polarization resolved anisotropic X-ray diffraction analysis of photochemical populations for the ultrafast time domain. This article is part of the theme issue 'Measurement of ultrafast electronic and structural dynamics with X-rays'.
RESUMEN
The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.
Asunto(s)
Cristalografía por Rayos X/métodos , Método de Montecarlo , Sincrotrones , beta-Lactamasas/metabolismoRESUMEN
X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.
Asunto(s)
Cristalografía por Rayos X/métodos , Sustancias Macromoleculares/química , Manejo de Especímenes/métodos , Cristalización , Desarrollo de Medicamentos/métodos , Electrones , Rayos Láser , Sustancias Macromoleculares/farmacología , Viscosidad/efectos de los fármacosRESUMEN
The multifarious injection chamber for molecular structure study (MICOSS) experimental system has been developed at the Pohang Accelerator Laboratory X-ray Free-Electron Laser for conducting serial femtosecond crystallography. This system comprises several instruments such as a dedicated sample chamber, sample injectors, sample environment diagnostic system and detector stage for convenient distance manipulation. Serial femtosecond crystallography experiments of lysozyme crystals have been conducted successfully. The diffraction peaks have reached to â¼1.8â Å resolution at the photon energy of 9.785â keV.