Diamond Light Source Ltd, Harwell Science & Innovation Campus.
Diamond Light Source Ltd, Harwell Science & Innovation Campus;
J Vis Exp. 2021 Apr 23(170). doi: 10.3791/62364.
Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 Å. To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.
长波长大分子晶体学（MX）利用硫、磷、钾、氯或钙等元素的反常散射特性，这些元素通常天然存在于大分子中。这使得蛋白质和核酸可以通过实验相变法直接解决结构问题，而无需额外的标记。为了消除该波长范围内 X 射线的显著空气吸收，这些实验在真空环境中进行。英国钻石光源的 I23 光束线是同类中的第一个同步加速器仪器，专为长波长范围内的 MX 实验而设计和优化，波长可达 5 Å。为了实现这一目标，一个大型真空容器将样品环境的所有末端站组件封闭在其中。为了在真空环境中储存和收集数据时保持样品处于低温状态，需要使用热传导性样品架。这有利于有效地去除热量，以确保样品冷却到约 50 K。本方案描述了在 I23 光束线上进行样品制备和转移到真空环境中的程序。确保在已经建立的大分子晶体学社区的实践和方法方面保持一致，将样品冷却到液氮温度可以在配备有标准 MX 工具的任何实验室环境中进行。低温储存和运输样品仅需要标准的商业上可用的设备。将低温冷却的晶体从液氮转移到真空末端站需要专门的设备。已经在内部开发了定制的样品处理工具和专用的低温传输系统（CTS）。使用此方案制备的样品收集的衍射数据显示出出色的合并统计数据，表明在整个过程中样品的质量没有变化。这为超出标准同步加速器光束线的波长范围内的真空 MX 提供了独特的机会。
