Improving high viscosity extrusion of microcrystals for time-resolved serial femtosecond crystallography at x-ray lasers

Daniel James, Tobias Weinert, Petr Skopintsev, Antonia Furrer, Dardan Gashi, Tomoyuki Tanaka, Eriko Nango, Przemyslaw Nogly, Joerg Standfuss

研究成果: Article査読

7 被引用数 (Scopus)

抄録

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

本文言語English
論文番号e59087
ジャーナルJournal of Visualized Experiments
2019
144
DOI
出版ステータスPublished - 2019 2
外部発表はい

ASJC Scopus subject areas

  • 神経科学(全般)
  • 化学工学(全般)
  • 生化学、遺伝学、分子生物学(全般)
  • 免疫学および微生物学(全般)

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