RESUMO
The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Conformação Proteica , Luz , Modelos Moleculares , Fatores de TempoRESUMO
The Adaptive Gain Integrating Pixel Detector (AGIPD) is an X-ray imager, custom designed for the European X-ray Free-Electron Laser (XFEL). It is a fast, low-noise integrating detector, with an adaptive gain amplifier per pixel. This has an equivalent noise of less than 1â keV when detecting single photons and, when switched into another gain state, a dynamic range of more than 104â photons of 12â keV. In burst mode the system is able to store 352 images while running at up to 6.5â MHz, which is compatible with the 4.5â MHz frame rate at the European XFEL. The AGIPD system was installed and commissioned in August 2017, and successfully used for the first experiments at the Single Particles, Clusters and Biomolecules (SPB) experimental station at the European XFEL since September 2017. This paper describes the principal components and performance parameters of the system.
RESUMO
Serial femtosecond crystallography is a rapidly developing method for determining the structure of biomolecules for samples which have proven challenging with conventional X-ray crystallography, such as for membrane proteins and microcrystals, or for time-resolved studies. The European XFEL, the first high repetition rate hard X-ray free electron laser, provides the ability to record diffraction data at more than an order of magnitude faster than previously achievable, putting increased demand on sample delivery and data processing. This work describes a publicly available serial femtosecond crystallography dataset collected at the SPB/SFX instrument at the European XFEL. This dataset contains information suitable for algorithmic development for detector calibration, image classification and structure determination, as well as testing and training for future users of the European XFEL and other XFELs.
RESUMO
Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
Assuntos
Cristalografia/instrumentação , Elétrons , Dispositivos Lab-On-A-Chip , Lasers , Aldeído Liases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , HidrodinâmicaRESUMO
We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.
Assuntos
Concanavalina A/química , Proteínas de Plantas/química , Urease/química , Cristalização , Cristalografia por Raios XRESUMO
The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength range suitable for atomic resolution structure determination. An outstanding but crucial question is whether the use of a pulse repetition rate nearly four orders of magnitude higher than previously possible results in unwanted structural changes due to either radiation damage or systematic effects on data quality. Here, separate structures from the first and subsequent pulses in the European XFEL pulse train were determined, showing that there is essentially no difference between structures determined from different pulses under currently available operating conditions at the European XFEL.
RESUMO
X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 µm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.