Nevertheless, they require handling of the crystals during loading, which can be problematic for samples that are sensitive to, for example, humidity changes. Clearly, high-viscosity injectors and fixed targets promote low sample consumption and are amenable to light-based pump–probe time-resolved experiments.
It is thus vital to consider which experimental design is most suitable for the sample and biology in question.
Running model made in comsol 5.1 in 5.2a serial#
As shown in Table 1, which summarizes the different serial crystallography experiments performed at monochromatic X-ray sources reported to date, data-collection times and sample consumption vary widely and depend greatly on the delivery method used and the nature of the protein-crystal slurry (crystal quality, concentration and monodispersity). Sample-delivery methods for SSX are not `one size fits all'. Liquid jets, such as those used to deliver samples at XFELs, are not suitable for millisecond crystallography owing to the very short residency time of the crystals in the X-ray-interaction region as a result of the fast fluid flow. For monochromatic X-ray diffraction work, these platforms have included raster-scanning or small-wedge data collection from small crystals mounted in micromeshes (Zander et al., 2015 ), the use of low-background fixed targets (Owen et al., 2017 Roedig et al., 2016 Zarrine-Afsar et al., 2012 Mueller et al., 2015 Huang et al., 2015 Coquelle et al., 2015 Baxter et al., 2016 Axford et al., 2016 Schubert et al., 2016 Doak et al., 2018 ), conveyor belts coupled to acoustic injectors (Roessler et al., 2013 ) or to liquid dispensers (Beyerlein et al., 2017 ), high-viscosity injectors (Weinert et al., 2017 Kovácsová et al., 2017 Sugahara et al., 2015 ), quartz capillaries (Stellato et al., 2014 ) and more recently microfluidic flow devices (Monteiro et al., 2019 ).
This interest has driven the development of novel and increasingly user-friendly sample-delivery methods (Yamamoto et al., 2017 Sui & Perry, 2017 ). However, the limited availability of XFEL time in conjunction with the continuous developments in hardware at synchrotrons, including newer faster detectors as well as bright microfocus beams, has resulted in increased interest of the structural biology community in serial millisecond crystallography. The serial approach is absolutely required at XFELs, where only one diffraction pattern is collected from the very short X-ray pulse (tens of femtoseconds) before the sample disintegrates as plasma (diffraction before destruction Chapman et al., 2011 Neutze et al., 2000 ). The sample refreshment also allows data to be collected at room temperature, eliminating any structural artifacts that arise during cryocooling (Fraser et al., 2011 ), as well as opening up the possibility of harnessing more information about protein dynamics.Īlthough multi-crystal experiments have been carried out in the past for very radiation-sensitive samples such as viruses (Abrescia et al., 2004 Ji et al., 2009 ), the high brilliance of third- and fourth-generation synchrotrons and X-ray free-electron laser (XFEL) sources has propelled the recent rapid development of serial crystallography. One of the main advantages of this technique is the low X-ray dose that is accumulated by the crystals during data collection, as fresh crystalline material is available for each new exposure (Ebrahim et al., 2019 Owen et al., 2017 ). Serial synchrotron crystallography (SSX) is a data-collection approach in which diffraction data are collected from low-millisecond X-ray exposures of protein microcrystals. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses.
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