X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs.

由于空间相干性，极高的峰值亮度和短的X射线脉冲持续时间的独特结合，X射线自由电子激光（XFEL）具有革新高分子结构生物学的潜力。最近出现的使用XFEL辐射的系列飞秒（fs）晶体学（SFX）方法通过破坏前衍射原理克服了与辐射损伤相关的传统晶体学的一些最大障碍。强大的fs XFEL脉冲可对难以结晶的生物大分子进行高分辨率的室温结构测定，同时开启了时间分辨结构研究的新纪元。在这里，我们回顾了仪器，样品输送，数据分析，结晶方法以及SFX在重要生物学问题上的应用方面的最新进展，