Hydroxyethyl cellulose matrix applied to serial crystallography

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.

Shortening injection matrix for serial crystallography

“…Based on their physical and chemical properties, sample delivery media can be categorized as either lipidic cubic phase (LCP), 15 oil-based, 16–18 or hydrogel-based. 17–21 Among them, LCP and grease, which have hydrophobic characteristics, provide the most stable injection streams and are the most widely used delivery media in SX experiments.…”

“…17–21 Among them, LCP and grease, which have hydrophobic characteristics, provide the most stable injection streams and are the most widely used delivery media in SX experiments. 15–18 LCP, which is usually used with monoolein, is widely applied in the process of membrane protein crystallization, and its crystallized membrane protein embedded in LCP can be used as a sample delivery medium. 15,23 The LCP delivery medium enables the sample to be delivered very steadily, even at flow rates of 0.001–0.3 μl/min.…”

“…15 However, the phase of LCP can be transformed into lamellar, hexagonal, or sponge, depending on the crystallization or experimental environment, and it is not suitable for use with high concentration of ammonium sulfate. 9,26 Oil-based grease matrices, such as mineral oil grease, 16 synthetic grease Super Lube, 17 and nuclear grade grease, 18 also provide stable injection streams and have been applied to various SFX experiments. However, because this material is usually in a semisolid state at room temperature, it is difficult to transfer it to a syringe or other devices, which can cause sample loss during sample preparation.…”

A guide to sample delivery systems for serial crystallography

“…However, because of the strong adhesion of HA, the nozzle is easily clogged. In 2017, Sugahara used an HEC matrix as the crystal carrier to obtain structures of soluble proteins at a resolution beyond 1.8 Å at room temperature. The HEC carrier can produce a stable and small‐diameter sample flow, and it is helpful to reduce sample consumption and background scatter.…”

“…However, because of the strong adhesion of HA, the nozzle is easily clogged. In 2017, Sugahara [75] used an HEC matrix as the crystal carrier to obtain structures of soluble proteins at a resolution beyond 1. 8 A at room temperature.…”