期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 181, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/63022
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资金
- US Department of Energy [DE-AC02-06CH11357]
- National Institute of General Medical Sciences of the National Institutes of Health [R24GM111072]
- Michigan Economic Development Corporation and Michigan Technology Tri-Corridor [085P1000817]
- University of Illinois at Chicago, National Institutes of Health [R01EY024363]
- National Science Foundation [MCB 2017274]
This article introduces a powerful method for conducting X-ray diffraction experiments at room temperature, which allows for high-throughput processing of hundreds to thousands of protein crystals without sample manipulation.
Biochemical reactions and biological processes can be best understood by demonstrating how proteins transition among their functional states. Since cryogenic temperatures are non-physiological and may prevent, deter, or even alter protein structural dynamics, a robust method for routine X-ray diffraction experiments at room temperature is highly desirable. The crystal-on-crystal device and its accompanying hardware and software used in this protocol are designed to enable in situ X-ray diffraction at room temperature for protein crystals of different sizes without any sample manipulation. Here we present the protocols for the key steps from device assembly, on-chip crystallization, optical scanning, crystal recognition to X-ray shot planning and automated data collection. Since this platform requires no crystal harvesting nor any other sample manipulation, hundreds to thousands of protein crystals grown on chip can be introduced into an X-ray beam in a programmable and high-throughput manner.
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