Abstract
Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.
Highlights
As structural biologists tackle ever more challenging systems, the development of efficient methods to deliver large quantities of crystals for X-ray diffraction studies is increasingly important
The structural information accessible from small or radiation-sensitive crystals may be extended through the application of femtosecond crystallography (FX), an emerging method that capitalizes on the extremely bright, short time-scale X-ray pulses produced by X-ray free-electron lasers (XFELs)
The process of sample exchange using automated mounting systems, which includes mounting a crystal, centering a crystal for data collection and dismounting the crystal, can take between 35 s and a few minutes. While this time scale may be suitable for experiments that require screening of at most a few hundred crystals, higher efficiency methods are critical for more challenging experiments at XFEL sources, where time is limited and in high demand
Summary
As structural biologists tackle ever more challenging systems, the development of efficient methods to deliver large quantities of crystals for X-ray diffraction studies is increasingly important. The process of sample exchange using automated mounting systems, which includes mounting a crystal, centering a crystal for data collection and dismounting the crystal, can take between 35 s and a few minutes While this time scale may be suitable for experiments that require screening of at most a few hundred crystals, higher efficiency methods are critical for more challenging experiments at XFEL sources, where time is limited and in high demand. We describe a simple, inexpensive high-density sample-mounting grid which enables efficient automated data collection from a large number of crystals that will only survive a few X-ray exposures The use of these grids to collect a complete, radiation-damage-free data set using myoglobin crystals at the Linear Coherent Light Source (LCLS) XFEL has been described previously (Cohen et al, 2014). To illustrate the techniques involved, we detail experiments in which crystals were grown on grids or loaded onto grids using liquidhandling robots for diffraction-quality screening experiments at LCLS-XPP
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