RESUMEN
In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a `sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.
Asunto(s)
Oxígeno , Sincrotrones , Cristalografía , Anaerobiosis , Cristalografía por Rayos X , Sustancias MacromolecularesRESUMEN
Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via light-weight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12â Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
RESUMEN
Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
Asunto(s)
Cristalografía por Rayos X/instrumentación , Sincrotrones , Diseño de Equipo , Laboratorios , Compuestos de Silicona , Suecia , TemperaturaRESUMEN
BioMAX is the first macromolecular crystallography beamline at the MAXâ IV Laboratory 3â GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20â µm × 5â µm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25â keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, an ISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAXâ IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAXâ IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1â µm × 1â µm beam focus and a flux up to 1015â photonsâ s-1 with main applications in serial crystallography, room-temperature structure determinations and time-resolved experiments.
RESUMEN
The interaction between single-walled carbon nanotubes and photosynthetic reaction centers purified from purple bacterium Rhodobacter sphaeroides R-26 has been investigated. Atomic force microscopy studies provide evidence that reaction center protein can be attached effectively to the nanotubes. The typical diameter of the nanotube is 1-4 nm and 15 +/- 2 nm without and with the reaction centers, respectively. Light-induced absorption change measurements indicate the stabilization of the P+(Q(A)Q(B))- charge pair, which is formed after single saturating light excitation after the attachment to nanotubes. The separation of light-induced charges is followed by slow reorganization of the protein structure. The stabilization effect of light-initiated charges by the carbon nanotubes opens a possible direction of several applications, the most promising being in energy conversion and storage devices.
Asunto(s)
Nanotubos de Carbono/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/metabolismo , Electroquímica , Cinética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Unión Proteica , Rhodobacter sphaeroides/químicaRESUMEN
A refined Fourier-transform method of analysis of interference patterns is presented. The refinements include a method of automatic background subtraction and a way of treating the problem of heterodyning. The method proves particularly useful for analysis of long sequences of interferograms.