Nylon mesh-based sample holder for fixed-target serial femtosecond crystallography

Abstract

Fixed-target serial femtosecond crystallography (FT-SFX) was an important advance in crystallography by dramatically reducing sample consumption, while maintaining the benefits of SFX for obtaining crystal structures at room temperature without radiation damage. Despite a number of advantages, preparation of a sample holder for the sample delivery in FT-SFX with the use of many crystals in a single mount at ambient temperature is challenging as it can be complicated and costly, and thus, development of an efficient sample holder is essential. In this study, we introduced a nylon mesh-based sample holder enclosed by a polyimide film. This sample holder can be rapidly manufactured using a commercially available nylon mesh with pores of a desired size at a low cost without challenging technology. Furthermore, this simple device is highly efficient in data acquisition. We performed FT-SFX using a nylon mesh-based sample holder and collected over 130,000 images on a single sample holder using a 30 Hz X-ray pulse for 1.2 h. We determined the crystal structures of lysozyme and glucose isomerase using the nylon mesh at 1.65 and 1.75 Å, respectively. The nylon mesh exposed to X-rays produced very low levels of background scattering at 3.75 and 4.30 Å, which are negligible for data analysis. Our method provides a simple and rapid but highly efficient way to deliver samples for FT-SFX.

Shortening injection matrix for serial crystallography

“…29,30 These metal binding sites of glucose isomerase are well-defined in the electron density map, and there was no negative fo-fc electron density map counted at 3 σ, indicating there was no significant radiation damage (Supplementary Figure S5). The glucose isomerase structures delivered in shortenings A and B had high similarity with previously reported glucose isomerase structures delivered using grease (PDB code 4W4Q) 31 and nylon mesh-based fixed target scanning (6IRK) 12 with a root mean squared deviation of 0.1594–0.2549 for Cα atoms (Supplementary Figure S6).…”

“…Glucose isomerase was supplied as a crystal suspension, and it was used directly for SMX experiments, as previously reported. 12 The lysozyme was crystallized using a previously reported procedure. 12 The crystal sizes of glucose isomerase and lysozyme were 30–40 μm and <60 μm, respectively.…”

“…5 Delivering the sample reliably not only reduces sample consumption but also enables data collection for accurate responses in time-resolved studies. 6 Among the various sample delivery methods, including liquid jet injector, 7 high viscosity injector, 8,9 fixed-target scanning, 10–12 and microfluidics, 13,14 sample delivery using a viscous medium with an injector or syringe is widely applied to serial femtosecond crystallography (SFX) or serial millisecond crystallography (SMX), which can successfully determine structure using an XFEL or synchrotron, respectivley. 15–22 The advantage of crystal delivery with a viscous delivery medium is the drastic reduction of sample consumption by lowering the flow rate of the sample loading from the injector or syringe.…”

Sample delivery using capillary and delivery medium for serial crystallography

“…Lysozyme crystals from a chicken white egg and glucose isomerase Streptomyces rubiginosus were prepared as previously reported. 16 The and glucose isomerase (20 μ l) was embedded in 5% (w/v) agarose (30 μ l) and 5% (w/v) gelatin (30 μ l), respectively, by mechanical mixing in a dual syringe setup. 19 Crystals embedded in the delivery medium were transferred into a 100 μ l syringe; this syringe was connected to the capillary-associated needle.…”

“…SX experiments require crystal samples to be delivered to the X-ray interaction point in a steady and stable manner. 12 Various sample delivery techniques such as injector-based methods, 13,14 fixed-target scanning, 15,16 electrospinning, 17 and methods employing microfluidic devices 18 have been developed, and their application has been successfully demonstrated in SX experiments. However, most of these methods require precisely fabricated sample delivery devices and are time consuming and expensive to perform.…”