Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

Abstract

The serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments.

Combination of an inject-and-transfer system for serial femtosecond crystallography

“…The fixed-target scanning method has advantages in that it consumes fewer crystal samples compared with the sample delivery method using an injector, minimizes physical damage on the crystal during sample delivery, and can be programmed so that the sample is delivered to the desired location (Hunter et al, 2014;Mueller et al, 2015;Roedig et al, 2017;Lee et al, 2019Lee et al, , 2020Nam et al, 2021). Although significant radiation damage has not yet been reported in crystal structures determined by the fixed-target scanning method, theoretically, radicals are generated after the X-rays have passed (Lee et al, 2019); therefore, diffusion of these radicals may affect the diffraction data of crystals collected later.…”

Processing of Multicrystal Diffraction Patterns in Macromolecular Crystallography Using Serial Crystallography Programs

“…Serial crystallography (SX) is an emerging X-ray crystallography technique that minimizes radiation damage and allows for the crystal structure to be determined at room temperature [11][12][13][14][15][16][17][18]. During SX measurements, a large number of crystals are exposed to the incident X-ray beam using a sample delivery system, such as an injector or syringe [19][20][21][22][23][24][25][26], fixed target [27][28][29], or microfluidic system [30,31], and the diffraction data are collected in a serial fashion. Since each crystal is not accurately delivered uniformly to the X-ray beam every time, the collected images included single-crystal diffraction data, multicrystal diffraction data, and diffraction-free data [32].…”

Combination of an inject-and-transfer system for serial femtosecond crystallography

“…The fixed target scanning method has advantages in that it consumes fewer crystal samples compared to the sample delivery method using an injector, minimizes physical impact on the crystal during Xray delivery, and can be programmed and delivered to the desired location [23,25,26,[33][34][35].…”

“…The fixed target scanning method has advantages in that it consumes fewer crystal samples compared to the sample delivery method using an injector, minimizes physical impact on the crystal during X-ray delivery, and can be programmed and delivered to the desired location [23, 25, 26, 33–35]. Although radiation damage has not yet been reported in the fixed target scanning method, theoretically, radicals are generated after XFEL is passed [26], so if they are diffused, they may affect the diffraction data of later collected crystals.…”