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Viscosity-adjustable grease matrices for serial nanocrystallography.

ABSTRACT: Serial femtosecond crystallography (SFX) has enabled determination of room temperature structures of proteins with minimum radiation damage. A highly viscous grease matrix acting as a crystal carrier for serial sample loading at a low flow rate of ~0.5??l?min-1 was introduced into the beam path of X-ray free-electron laser. This matrix makes it possible to determine the protein structure with a sample consumption of less than 1?mg of the protein. The viscosity of the matrix is an important factor in maintaining a continuous and stable sample column from a nozzle of a high viscosity micro-extrusion injector for serial sample loading. Using conventional commercial grease (an oil-based, viscous agent) with insufficient control of viscosity in a matrix often gives an unexpectedly low viscosity, providing an unstable sample stream, with effects such as curling of the stream. Adjustment of the grease viscosity is extremely difficult since the commercial grease contains unknown compounds, which may act as unexpected inhibitors of proteins. This study introduces two novel grease matrix carriers comprising known compounds with a viscosity higher than that of conventional greases, to determine the proteinase K structure from nano-/microcrystals.

SUBMITTER: Sugahara M

PROVIDER: S-EPMC6987181 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Viscosity-adjustable grease matrices for serial nanocrystallography.

Sugahara Michihiro M Motomura Koji K Suzuki Mamoru M Masuda Tetsuya T Joti Yasumasa Y Numata Keiji K Tono Kensuke K Yabashi Makina M Ishikawa Tetsuya T

Scientific reports 20200128 1

Serial femtosecond crystallography (SFX) has enabled determination of room temperature structures of proteins with minimum radiation damage. A highly viscous grease matrix acting as a crystal carrier for serial sample loading at a low flow rate of ~0.5 μl min<sup>-1</sup> was introduced into the beam path of X-ray free-electron laser. This matrix makes it possible to determine the protein structure with a sample consumption of less than 1 mg of the protein. The viscosity of the matrix is an impo ...[more]

PMID: 31992735

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Project description: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.

Dataset Information

Viscosity-adjustable grease matrices for serial nanocrystallography.

Publications

Viscosity-adjustable grease matrices for serial nanocrystallography.

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