Viscous hydrophilic injection matrices for serial crystallography.

R Bruce Doak,Gabriela Kovácsová,Marie Luise Grünbein,Thomas R M Barends,Joachim Heberle,Marco Kloos,Ramona Schlesinger,Ilme Schlichting,Wolfgang Kabsch,Robert L Shoeman

doi:10.1107/s2052252517005140

R Bruce Doak, Gabriela Kovácsová + Show 8 more

Open Access

https://doi.org/10.1107/s2052252517005140

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Abstract

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates - gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro-gels as viscous injection matrices is described, namely sodium carb-oxy-methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements.

Highlights

X-ray free-electron lasers (XFELs) provide coherent X-ray pulses of femtosecond duration and a peak brilliance that exceeds that of third-generation synchrotron sources by nine orders of magnitude
This is frequently achieved using either a GDVN injector (Weierstall et al, 2012) that produces a fast liquid microjet of protein crystals in their mother liquor, or an high-viscosity extrusion (HVE) injector (Weierstall et al, 2014; Botha et al, 2015) that slowly extrudes a stream of crystals embedded in a viscous medium
Injection properties were optically judged as good (+) or very good (++) depending on the stability and viscosity of the extruded stream

Summary

Introduction

X-ray free-electron lasers (XFELs) provide coherent X-ray pulses of femtosecond duration and a peak brilliance that exceeds that of third-generation synchrotron sources by nine orders of magnitude. These unique X-ray beam properties significantly extend the possibilities of macromolecular crystallography by ‘outrunning’ most radiation damage effects (Chapman et al, 2011; Boutet et al, 2012). They thereby reduce the required crystal size for diffraction data collection, which in turn facilitates reaction initiation for time-resolved experiments. Complete coverage of reciprocal space is typically obtained by collecting a large number of diffraction patterns of randomly oriented (micro)crystals, followed by merging of many partial intensities (Kirian et al, 2010; White et al, 2012; Hattne et al, 2015; Kabsch, 2014)

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