Fixed-Target Serial Synchrotron Crystallography Using Nylon Mesh and Enclosed Film-Based Sample Holder

Abstract

Serial crystallography (SX) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment.

Room-Temperature Structure of Xylitol-Bound Glucose Isomerase by Serial Crystallography: Xylitol Binding in the M1 Site Induces Release of Metal Bound in the M2 Site

“…Raster scans were performed at a 50 μm scan interval in both vertical and horizontal directions. The data collection strategy was similar to that reported previously [ 33 ]. The crystals were exposed to the X-ray beam for 100 ms, with an oscillation of 0.007° at each raster scan point.…”

Molecular Dynamics—From Small Molecules to Macromolecules

“…Additionally, time-resolved SFX studies using an optical laser or mix-and-inject technique contribute to the making movies with high resolution molecular dynamics [ 15 , 27 ]. Moreover, due to advances in X-ray-focusing technology and detectors, time-resolved serial millisecond crystallography (SMX) studies as well as general serial synchrotron crystallography (SSX) are being performed at the existing synchrotron facilities [ 28 , 29 , 30 ]. Recently, the analysis of large complex structures using Cryo-EM technology has been in the spotlight [ 31 ], and the time-resolved EM technique is expected to be useful in the future for obtaining molecular dynamics information [ 32 ].…”

Approach of Serial Crystallography

“…As an alternative to this technique, serial millisecond crystallography (SMX) or serial synchrotron crystallography (SSX) based on synchrotron X-rays has been recently used [28][29][30][31][32]. In order to perform the SX using synchrotron X-rays, it is important not only to provide a high photon flux at the sample position but also to enable fast readout by a detector [33]. Currently, many macromolecule crystallography beamlines at synchrotrons have achieved X-ray focusing using optics (e.g., K-B mirror) by up to a few microns and high-speed readout detectors are commercially available.…”

“…Moreover, a sample delivery method is required to continuously and reliably deliver a large number of crystal samples to the X-ray interaction point for diffraction data collection [34,35]. Various sample delivery methods such as the use of injectors [36,37], syringes [38,39], injection with carrier matrix [35,[37][38][39][40][41][42][43][44], fixed-target scanning [33,[45][46][47][48], microfluidics [49][50][51], and conveyor belt approaches [52,53] are developed. These techniques are used to carry out the SX experiment and crystal structure can be determined successfully.…”

“…In the early stages of SX research, sample delivery and data collection strategies based on injectors were common; however, now diverse sample delivery methods and data collection strategies have been reported [26,[33][34][35]59,60]. Although the SX research approach used by each facility cannot be applied in the same manner to all other facilities, reporting and sharing of SX approaches followed in each research institute and facility will provide new insights to the SX community in the future and provide useful information for development of a universal system that can be easily accessed by general researchers.…”