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The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.


Using the two matrix carriers, Super Lube grease and hyaluronic acid, we successfully collected the data sets for proteinase K and lysozyme at 2.3-Å resolution (Table 1). A 12% (w/v) hyaluronic acid matrix prepared by mixing 24% (w/v) hyaluronic acid aqueous solution with an equal volume of the supernatant solution in the crystal suspension solution was used for the lysozyme crystals. Optimizing the hyaluronic acid solution buffer is important to prevent any damage to the crystals. We have observed that before adding protein crystals it is essential to mix the hyaluronic acid aqueous solution with the supernatant solution or the crystal harvest solution, which helps to avoid osmotic shock to the crystals when mixing with the medium. The hyaluronic acid solution was saturated with the supernatant solution or the crystal harvest solution, and then protein crystals were added, which helps to avoid potential osmotic shock to the crystals. The unit-cell axes of the lysozyme crystals for the grease matrix were slightly shorter than those for the hyaluronic acid matrix (Table 1). Dehydration of protein crystals might have been induced during the sample preparation process of the water-free grease matrix. In such cases, a water-based medium can be helpful for preventing the contraction of the unit cell in the SFX experiments.


Recently, using the grease matrix technique, Yamashita and coworkers have demonstrated a single isomorphous replacement with anomalous scattering (SIRAS) phasing for Hg-derivatized luciferin-regenerating enzyme17. In addition, we have successfully determined the structure of native lysozyme with single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine18. One of the major challenges for phasing in SFX is to improve the signal-to-noise ratio. In this study, we could observe a weak anomalous scattering signal from the calcium atom in the proteinase K structures (Fig. 2). The anomalous difference Fourier maps showed that the signal from the calcium atom is stronger with the hyaluronic acid matrix than with the grease matrix. Furthermore, in the crystal structure for the hyaluronic acid matrix, we could observe anomalous signal from sulfur atoms (e.g. the sulfur atom of Cys178, Fig. 2b), which was not discernible when using the grease matrix. This technique using the matrices with low background scattering noise will contribute significantly to measuring weak anomalous signals for de novo phasing from SFX data.


M.Sugahara. introduced the oil-free hyaluronic acid matrix extrusion scheme, M.Suzuki, T.M., S.Inoue, F.Y., E.N., R.T., C.S. and M.Sugahara performed the data collection, M.Sugahara. and M.Suzuki performed data processing and solved the structure, M.Sugahara, E.N. and K.N. prepared the microcrystals, T.N. and O.N. developed the data processing pipeline and performed the background scattering data analysis, K.T., Y.J., T.K., T.H. and M.Y. developed the DAPHNIS and detectors, M.Sugahara, C.S., M.Suzuki, T.M., S.Inoue and T.N. wrote the manuscript with input from all of the coauthors and S.Iwata coordinated the project.


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