Viscous hydrophilic injection matrices for serial crystallography.
Viscous hydrophilic injection matrices for serial crystallography.
复制标题
粘性亲水性注射矩阵，用于连续晶体学。
DOI：
10.1107/s2052252517005140
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发表时间：
2017-07-01
期刊：
影响因子：
3.9
通讯作者：
Schlichting I
中科院分区：
文献类型：
--
作者：
Kovácsová G;Grünbein ML;Kloos M;Barends TRM;Schlesinger R;Heberle J;Kabsch W;Shoeman RL;Doak RB;Schlichting I
高粘度挤压注射是在同步加速器和XFELs进行串行测量的高通量晶体输送的有效手段。描述了用于晶体包埋的水凝胶，它与广泛的结晶沉淀剂兼容，产生低x射线背景，并提供适合时间分辨测量的非常稳定的流。同步加速器和x射线自由电子激光（XFEL）源的串行（飞秒）晶体学将吸收的辐射剂量分布在用于数据收集的所有晶体上，因此可以测量辐射损伤易发系统，包括使用微晶体进行室温测量。序列晶体学依赖于x射线曝光后快速有效的晶体交换，这可以通过各种方法实现，包括各种注射技术。后者的流量差异很大——基于气体动态虚拟喷嘴的喷射器提供非常薄的快速流动射流，而高粘度挤出喷射器产生的射流更厚，流速低2到3个数量级。高粘度挤压导致更低的样品消耗，因为它的样品输送速度与典型的XFEL重复率和同步加速器源的数据采集率相当。脂质立方相（LCP）是用于介膜蛋白结晶的一种明显的粘性注射介质。然而，LCP与许多结晶条件的相容性有限。虽然文献中已经描述了一些其他粘性介质，但仍然需要确定用于晶体嵌入的其他注入介质。关键属性是可靠的注射特性和广泛的化学相容性，以适应样品的异质和敏感的蛋白质晶体。本文描述了两种新型水凝胶作为粘性注射基质的使用，即碳水化合物-氧-甲基纤维素钠和热可逆嵌段聚合物Pluronic F-127。两者都能适应各种结晶条件，并产生可接受的x射线背景。对挤压流的稳定性和速度进行了分析，并测量了挤压流速度与流量的关系。与先前表征的注射介质相比，这两种新基质提供非常稳定的可调流，适合于时间分辨测量。
High-viscosity extrusion injection is an efficient means for high-throughput crystal delivery for serial measurements at synchrotrons and XFELs. Hydrogels for crystal embedding are described that are compatible with a wide range of crystallization precipitants, result in low X-ray background and afford very stable streams suitable for time-resolved measurements. Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydrogels as viscous injection matrices is described, namely sodium carboxymethyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.