Hydroxyethyl cellulose matrix applied to serial crystallography

Michihiro Sugahara,Osamu Nureki,Rie Tanaka,Tetsuya Masuda,Kensuke Tono,Makina Yabashi,Takashi Kameshima,Fumiaki Yumoto,Shigeyuki Inoue,Eriko Nango,Eiichi Mizohata,Changyong Song,Takaki Hatsui,So Iwata,Keiji Numata,Toru Nakatsu,Mamoru Suzuki,Yasumasa Joti,Takanori Nakane

doi:10.1038/s41598-017-00761-0

Abstract

Serial femtosecond crystallography (SFX) allows structures of proteins to be determined at room temperature with minimal radiation damage. A highly viscous matrix acts as a crystal carrier for serial sample loading at a low flow rate that enables the determination of the structure, while requiring consumption of less than 1 mg of the sample. However, a reliable and versatile carrier matrix for a wide variety of protein samples is still elusive. Here we introduce a hydroxyethyl cellulose-matrix carrier, to determine the structure of three proteins. The de novo structure determination of proteinase K from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of the praseodymium atom was demonstrated using 3,000 diffraction images.

Highlights

Background intensity determinationThe background intensities from Super Lube synthetic grease, Super Lube nuclear grease and hydroxyethyl cellulose through a 100-μm-i.d. nozzle at 1.77 Å and that from LCP14 through a 75-μm-i.d. nozzle at 1.61 Å were determined by a procedure similar to that used in Conrad et al.[23]
We found that 3,000 indexed images were sufficient for single-wavelength anomalous diffraction (SAD) phasing of proteinase K crystals
Our cellulose matrix with low background scattering noise is compatible with the accurate measurement of weak anomalous signals essential for de novo phasing from SFX data

Summary

Introduction

Background intensity determinationThe background intensities from Super Lube synthetic grease, Super Lube nuclear grease and hydroxyethyl cellulose through a 100-μm-i.d. nozzle at 1.77 Å and that from LCP14 through a 75-μm-i.d. nozzle at 1.61 Å were determined by a procedure similar to that used in Conrad et al.[23]. For data collection from small crystals (~1 μm), at atomic resolution, and de novo phasing with weak anomalous signals, a crystal carrier with low background scattering is essential to improve the signal-to-noise ratio[23]. We demonstrate the cellulose matrix as a protein carrier for SFX using small and large sized crystals (1 × 1 × 1 to 20 × 20 × 30 μm).

Results

Conclusion