A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography

Abstract

Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.

A snapshot love story: what serial crystallography has done and will do for us

“…Conveyor-belt-based hybrid methods (Butryn et al, 2021;Zielinski et al, 2022) or microfluidic sample-delivery systems (Ghosh et al, 2023) are compatible with future high-throughput approaches (Fig. 3) and time-resolved measurements, consume relatively small amounts of sample (comparable to fixedtarget chips) and are less prone to clogging than liquid jets.…”

Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron

“…Despite its exceptional potential to advance the field of structural enzymology, the method has not been widely applied because of its dependence on the availability of limited XFEL beamtime. MISC stands in contrast with other XFEL-pioneered methods, such as serial crystallography, which have been successfully modified for synchrotron sources [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] . The growth of synchrotron serial crystallography (SSX) reflects the increased interest in using synchrotrons for these experiments, motivated in part by the more abundant beamtime for structural biology at these facilities.…”

Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes

“…The development of fourth-generation storage-ring light sources (Eriksson et al, 2014) has enabled X-ray beams with high brilliance and micrometre-scale focusing; combined with advances in fastframe-rate X-ray detectors (Leonarski et al, 2020), dedicated SSX beamline endstations are now available for users. Current sample-delivery methods for SSX broadly fall into one of two categories: fixed-target methods, where crystal slurries are deposited onto a substrate that is rastered across the beam (Sherrell et al, 2015(Sherrell et al, , 2022Schulz et al, 2018;Zielinski et al, 2022), and flow systems, where crystals suspended in a medium are directly passed through the beam (Botha et al, 2015;Shilova et al, 2020;Nam, 2020;Knos ˇka et al, 2020;Ghosh et al, 2023). Still, challenges remain, from the amount of sample used to heating of the system, clogging of capillaries, burning of the material and sample sensitivity.…”