Polyacrylamide injection matrix for serial femtosecond crystallography

Abstract

Serial femtosecond crystallography (SFX) provides opportunities to observe the dynamics of macromolecules without causing radiation damage at room temperature. Although SFX provides a biologically more reliable crystal structure than provided by the existing synchrotron sources, there are limitations due to the consumption of many crystal samples. A viscous medium as a carrier matrix reduces the flow rate of the crystal sample from the injector, thereby dramatically reducing sample consumption. However, the currently available media cannot be applied to specific crystal samples owing to reactions between the viscous medium and crystal sample. The discovery and characterisation of a new delivery medium for SFX can further expand its use. Herein, we report the preparation of a polyacrylamide (PAM) injection matrix to determine the crystal structure with an X-ray free-electron laser. We obtained 11,936 and 22,213 indexed images using 0.5 mg lysozyme and 1.0 mg thermolysin, respectively. We determined the crystal structures of lysozyme and thermolysin delivered in PAM at 1.7 Å and 1.8 Å resolutions. The maximum background scattering from PAM was lower than monoolein, a commonly used viscous medium. Our results show that PAM can be used as a sample delivery media in SFX studies.

Shortening injection matrix for serial crystallography

“…The X-ray exposed sample delivery media generate background scattering, which can affect the signal-to-noise (SNR) ratio during data processing. 19,21 The background scattering from the delivery medium is one of the most important criteria for delivery medium selection for SX experiments. 22 The analyses of the background scattering of shortenings A and B were performed with the LCP delivery medium consisting of 60% (v/v) monoolein.…”

“…34 The hit images were indexed and merged using the CrystFEL program. 35 The phase problem of the lysozyme and glucose isomerase was solved by molecule replacement using the Phaser-MR in PHENIX 36 , with the crystal structures of lysozyme (PDB code 6IG6) 21 and glucose isomerase (PDB code 5ZYD) 30 used as the search models. The model building and refinement were conducted using Coot 37 and Phenix.refinement in PHENIX, respectively.…”

“…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. 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.…”

Sample delivery using capillary and delivery medium for serial crystallography

“…The diffraction patterns were filtered and processed using the Cheetah 27 and CrystFEL 28 programs, respectively. Molecular replacement was performed using Phaser-MR 29 with the crystal structure of lysozyme (PDB code 6IG6) 21 or glucose isomerase (5ZYE) 30 as the search modes. The model was established and its structure was refined using the COOT 31 and PHENIX 32 programs, respectively.…”

“…14,20 Meanwhile, sample delivery using an injector with a viscous delivery medium can drastically reduce the sample consumption at low flow rates. 19,21 However, the viscosity of the delivery medium varies depending on the crystallization solution and experimental environment; therefore, considerable effort is still required to maintain a stable injection stream. In contrast, a method of delivering a crystal suspension using a capillary was introduced previously.…”