Mix-and-extrude: high-viscosity sample injection towards time-resolved protein crystallography

Abstract

Time-resolved crystallography enables the visualization of protein molecular motion during a reaction. Although light is often used to initiate reactions in time-resolved crystallography, only a small number of proteins can be activated by light. However, many biological reactions can be triggered by the interaction between proteins and ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D-printed microchannels in conjunction with a micronozzle. The diffusive mixing enables the study of the dynamics of samples in viscous media. The device design allows mixing of the ligands and protein crystals in 2 to 20 s. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within lipidic cubic phase, can be mixed efficiently. The potential of this approach for time-resolved membrane protein crystallography to support the development of new drugs is discussed.

Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers

“…56 , 1038 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution (CC BY) license]; 29 (d) 2PP fabrication-based flat jet nozzle [Reproduced with permission from P. E. Konold et al , IUCrJ 10 , 662 (2023). Copyright 2023 Authors, licensed under a Creative Commons Attribution (CC BY) license].…”

“…These abilities greatly expand the possible device geometries, including helical mixers in a modular configuration, 28 asymmetric liquid outlets, 27 and high-viscosity sample injectors. 29 In direct laser writing (DLW) method, the printing time of two-photon printers scales with the total volume written. Because of this, the usual practice is to minimize the printed volume of two-photon devices to reduce the writing time and allow fabrication of multiple devices within reasonable times, i.e., seconds to minutes.…”

Structural Dynamics of Metalloproteins and Redox Enzymology with Mix-and-Inject Time-Resolved Serial Femtosecond Crystallography

“…Notably, based on currently available mixing delivery systems, ,,,,− the above-mentioned discussion holds true only for the MM kinetics of a standard single-substrate enzymatic reaction (Figure b). This approach is not applicable when considering enzymatic reactions involving two or more substrates, which is the case for many enzymes, such as those following the ping-pong bibi (double-displacement) or sequential mechanism. , One classical example of such enzymes is the heme peroxidase superfamily, which includes dye-decolorizing peroxidases (DyPs) and adopts a double-substrate ping-pong kinetic model .…”

“…36,149 We recently succeeded in developing a mixing-HVE device that can achieve mixing times of 2 to 20 s, and this was demonstrated via a fluorescence quenching experiment on iq-mEmerald fluorescent protein microcrystals. 123 It is possible to use mixing-HVE devices with various viscous materials, such as grease or Vaseline, 150−152 agarose, 153 hydroxyethyl cellulose, 154 and lards, 155 which can cover a wide range of different metalloenzymes and membrane proteins without compromising their diffraction quality. This approach can serve as a complementary mixing device for longer delay times within a single facility, allowing us to cover delay times from a few milliseconds to several seconds; however, it is still tedious to run experiments that require a wide range of delay times (e.g., from microseconds to milliseconds), for example, running both interaction regions of the SPB/SFX instrument within a single dedicated beamtime.…”

Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE

“…Second, liquid phase injectors, such as grease injectors, offer low sample consumption and high hit rates. 20,21 However, grease injectors are not suitable for MHz XFELs due to their slower delivery speeds and inability to refresh the sample volume. An alternative liquid target is the gas dynamic virtual nozzle (GDVN), which is the only method at present capable of delivering samples at >100 Hz repetition rates.…”