High-viscosity sample-injection device for serial femtosecond crystallography at atmospheric pressure.

Yoshiaki Shimazu,So Iwata,Kensuke Tono,Michihiro Sugahara,R Bruce Doak,Tatsuro Shimamura,Rie Tanaka,Yasuaki Yamanaka,Eriko Nango,Kanako Terakado Kimura,Chihiro Mori,Makina Yabashi,Takanori Nakane,Takaaki Fujiwara,Tomoyuki Tanaka

doi:10.1107/s1600576719012846

Abstract

A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump-probe techniques.

Highlights

Serial femtosecond crystallography (SFX) is a recently developed technique for determining protein crystal structures using X-ray free-electron lasers (XFELs) (Chapman et al, 2011; Boutet et al, 2012)
A total of 190 041 diffraction patterns were collected from ca 30 ml of the A2A adenosine receptor (A2AAR) crystals embedded in lipidic cubic phase (LCP), of which 10 816 were identified as hit images, corresponding to an average hit rate of 5.7%
The sample stream was chopped by the XFEL beam during data collection, the suction device and a co-flowing sheath gas delivered samples to the interaction point without coiling up

Summary

Introduction

Serial femtosecond crystallography (SFX) is a recently developed technique for determining protein crystal structures using X-ray free-electron lasers (XFELs) (Chapman et al, 2011; Boutet et al, 2012). Whereas conventional X-ray diffraction data collection using synchrotron sources requires measurements at cryogenic temperatures to prevent radiation damage, SFX allows data collection at room temperature because intense femtosecond XFEL pulses afford diffraction patterns from protein microcrystals before the onset of radiation damage (Neutze et al, 2000). Given that microcrystals are destroyed by a single XFEL pulse, replenishment of intact crystals is imperative for data collection in SFX. The crystal concentration governs the probability of the XFEL pulse hitting crystals in the carrier medium. A high crystal concentration causes clogging of the injector nozzle.

Results

Discussion

Conclusion