{"title":"Tracking Protein Motions using Serial Femtosecond Crystallography with X-Ray Free-Electron Laser","authors":"Eiichi Mizohata, Eriko Nango, Takehiko Tosha, So Iwata, Minoru Kubo","doi":"10.1002/cpz1.70212","DOIUrl":null,"url":null,"abstract":"<p>Since the birth of biochemistry, researchers have investigated the structure–function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Microcrystallization of lysozyme</p><p><b>Basic Protocol 2</b>: Microcrystallization of copper-containing nitrite reductase</p><p><b>Basic Protocol 3</b>: Time-resolved serial femtosecond crystallography</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 9","pages":""},"PeriodicalIF":2.2000,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70212","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.70212","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Since the birth of biochemistry, researchers have investigated the structure–function relationship of a wide variety of proteins. However, until recently, when X-ray free-electron lasers (XFELs) became available, it was not possible to visualize the motion of proteins from moment to moment with excellent temporal and spatial resolution. Here, we introduce practical methods to visualize protein motions at room temperature using serial femtosecond crystallography (SFX) using XFELs. With the development of this technology, it will be possible to visualize the entire reaction mechanism of many proteins in the future. We first outline a streamlined microcrystallization workflow for hen egg-white lysozyme, enabling rapid detector calibration and data-collection optimization. Next, we present a rotational seeding approach refined on copper-containing nitrite reductase that yields homogeneous microcrystals suitable for high-resolution SFX and readily adaptable to other challenging targets. Finally, we describe a time-resolved strategy combining microcrystals of fungal nitric-oxide reductase with photolabile caged substrates and synchronized UV triggering, capturing catalytic intermediates on the millisecond timescale. Together, these procedures enable investigators to progress from preparing samples to capturing dynamic structural snapshots. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1: Microcrystallization of lysozyme
Basic Protocol 2: Microcrystallization of copper-containing nitrite reductase
Basic Protocol 3: Time-resolved serial femtosecond crystallography
用x射线自由电子激光连续飞秒晶体学跟踪蛋白质运动。
自生物化学诞生以来,研究人员已经研究了各种蛋白质的结构-功能关系。然而,直到最近,当x射线自由电子激光器(XFELs)可用时,它不可能以优异的时间和空间分辨率可视化蛋白质的运动。在这里,我们介绍了在室温下使用XFELs的串行飞秒晶体学(SFX)可视化蛋白质运动的实用方法。随着这项技术的发展,未来将有可能可视化许多蛋白质的整个反应机制。我们首先概述了一种简化的蛋清溶菌酶微结晶工作流程,使检测器快速校准和数据收集优化成为可能。接下来,我们提出了一种基于含铜亚硝酸盐还原酶的旋转播种方法,该方法可产生适合高分辨率SFX的均匀微晶体,并易于适应其他具有挑战性的目标。最后,我们描述了一种时间解决策略,将真菌一氧化氮还原酶微晶体与光稳定的笼状底物和同步紫外线触发相结合,在毫秒时间尺度上捕获催化中间体。总之,这些程序使调查人员能够从准备样品到捕获动态结构快照。©2025作者。Wiley期刊有限责任公司发表的当前协议。基本协议1:溶菌酶微结晶基本协议2:含铜亚硝酸盐还原酶微结晶基本协议3:时间分辨串行飞秒晶体学。
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