Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.
Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.
复制标题
DOI：
10.1016/j.bpr.2022.100081
复制
发表时间：
2022-12-14
期刊：
影响因子：
--
通讯作者：
Ros, Alexandra
中科院分区：
文献类型：
--
作者：
Sonker, Mukul;Doppler, Diandra;Egatz-Gomez, Ana;Zaare, Sahba;Rabbani, Mohammad T;Manna, Abhik;Cruz Villarreal, Jorvani;Nelson, Garrett;Ketawala, Gihan K;Karpos, Konstantinos;Alvarez, Roberto C;Nazari, Reza;Thifault, Darren;Jernigan, Rebecca;Oberthur, Dominik;Han, Huijong;Sierra, Raymond;Hunter, Mark S;Batyuk, Alexander;Kupitz, Christopher J;Sublett, Robert E;Poitevin, Frederic;Lisova, Stella;Mariani, Valerio;Tolstikova, Alexandra;Boutet, Sebastien;Messerschmidt, Marc;Meza-Aguilar, J Domingo;Fromme, Raimund;Martin-Garcia, Jose M;Botha, Sabine;Fromme, Petra;Grant, Thomas D;Kirian, Richard A;Ros, Alexandra
随着X射线自由电子激光（XFEL）的发展，连续飞秒晶体学（SFX）已经能够对具有挑战性的蛋白质（如膜蛋白复合物）进行静态和动态结构测定。在具有XFEL的SFX中，晶体通常在与单个XFEL脉冲相互作用后被破坏。因此，必须将数千个新晶体依次引入X射线束中以收集完整的数据集。由于任何SFX实验的连续性质，由于所有当前XFEL的固有脉冲性质，在X射线脉冲之间的“关断时间”期间递送到X射线束的高达99%的样品被浪费。为了解决这一主要问题的大，往往限制样品消耗，我们报告的改进，革命性的样品保存方法，兼容所有当前的XFEL。我们先前报道了与气体动态虚拟喷嘴（GDVN）耦合的3D打印注射装置，其能够生成包含由不混溶油相分割的液滴的样品，用于将载有晶体的液滴喷射到XFEL的路径中。在这里，我们通过包括诱导电润湿效应的金属电极来进一步改进器件设计，用于改进对液滴产生频率的控制，以通过采用电反馈机制来刺激液滴释放以匹配XFEL重复率。我们报告的改进，在这种电触发的分段流动的方法与连续GDVN注射使用的溶菌酶和3-脱氧-D-甘露-辛酮糖酸8-磷酸合酶的微晶相比，样品保存和报告的分段流动的方法，样品注射应用在大分子飞秒晶体仪器在线性相干光源的第一次。
With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its “off-time” between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.