Fixed-target serial oscillation crystallography at room temperature

Abstract

A fixed-target approach to high-throughput room-temperature serial synchrotron crystallography with oscillation is described. Patterned silicon chips with microwells provide high crystal-loading density with an extremely high hit rate. The microfocus, undulator-fed beamline at CHESS, which has compound refractive optics and a fast-framing detector, was built and optimized for this experiment. The high-throughput oscillation method described here collects 1–5° of data per crystal at room temperature with fast (10° s−1) oscillation rates and translation times, giving a crystal-data collection rate of 2.5 Hz. Partial datasets collected by the oscillation method at a storage-ring source provide more complete data per crystal than still images, dramatically lowering the total number of crystals needed for a complete dataset suitable for structure solution and refinement – up to two orders of magnitude fewer being required. Thus, this method is particularly well suited to instances where crystal quantities are low. It is demonstrated, through comparison of first and last oscillation images of two systems, that dose and the effects of radiation damage can be minimized through fast rotation and low angular sweeps for each crystal.

Macromolecular room temperature crystallography

“…Ongoing work addresses problems with clogging and flow-speed of microfluidics, bubble generation, viscous stream focusing, deposition of varied-sized crystals, and sample consumption to enable the routine, uninterrupted collection of serial crystallography data. Popular fixed-target approaches include in situ deposition of crystal-containing drops on a humidity-controlled hit-and-return chip (Baxter et al ., 2016; Mehrabi et al ., 2019 b ; Mehrabi et al ., 2020), sandwiched between mylar, kapton, or cyclic-olefin-copolymer foils (Schubert et al ., 2016; Broecker et al ., 2018; Doak et al ., 2018; Feiler et al ., 2019; Wierman et al ., 2019), on micropatterned silicon chips (Mueller et al ., 2015; Roedig et al ., 2016; Meents et al ., 2017; Owen et al ., 2017; Ebrahim et al ., 2019 a ) or transported into the beam by a conveyor-belt like tape drive (Roessler et al ., 2013; Beyerlein et al ., 2017). Another option is to directly grow crystals on an in situ micropatterned silicon chip.…”

Evaluation of the data-collection strategy for room-temperature micro-crystallography studied by serial synchrotron rotation crystallography combined with the humid air and glue-coating method

“…Our results obtained at CT demonstrated that rotation is effective for efficient data collection in SSX and we called this method serial synchrotron rotation crystallography (SS-ROX; Hasegawa et al, 2017). Meanwhile, Wierman and coworkers reported the perfor-mance of serial oscillation crystallography with a fixed target, which demonstrated that a larger oscillation wedge decreased the total number of crystals needed to complete a data set (Wierman et al, 2019).…”

“…The usefulness of SFX has been demonstrated particularly well in a time-resolved structural study (Schmidt, 2019). The success of SFX led to the development of synchrotron serial crystallography (SSX) using either a continuous flow of a crystal suspension (see, for example, Stellato et al, 2014;Botha et al, 2015;Nogly et al, 2015;Martin-Garcia et al, 2017;Weinert et al, 2017) or a fixed-target method (see, for example, Gati et al, 2014;Coquelle et al, 2015;Owen et al, 2017;Wierman et al, 2019). Even though radiation damage must be taken into consideration, the feasibility of SSX has been demonstrated and it has successfully been applied to time-resolved structure analysis (Schulz et al, 2018;Weinert et al, 2019;Aumonier et al, 2020).…”

“…In the case of an injector-based method, samples are kept in the mother solution or in highviscosity media. In the case of a fixed-target method, two approaches are possible: one is sealing the sample holder with a polymer film (Owen et al, 2017;Wierman et al, 2019) and the other is the use of a humidifier (Roedig et al, 2016;Tolstikova et al, 2019).…”

An automated platform for in situ serial crystallography at room temperature

“…However, these delivery systems often suffer from a deficient hit rate and diminishingly minute yield, two metrics defined differently to measure the economy of crystal use (Ren et al, 2018), which severely limits their applicability to many biological systems. To address this challenge, a variety of fixed-target solutions have been proposed for serial crystallography (Baxter et al, 2016;Dhouib et al, 2009;Kisselman et al, 2011;le Maire et al, 2011;Perry et al, 2013;Wierman et al, 2019) and in situ diffraction (Axford et al, 2012;Bingel-Erlenmeyer et al, 2011;Sanchez-Weatherby et al, 2019), among which, we first proposed in situ serial Laue diffraction (Perry et al, 2014). However, implementation of an automated and robust platform for high-throughput serial data acquisition remains a challenging task for large-scale operation.…”