Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography

Abstract

Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.

Microfluidic rotating-target device capable of three-degrees-of-freedom motion for efficient in situ serial synchrotron crystallography

“…A number of sample delivery systems which can be categorized as liquid jet technology and fixed-target technology as well as hybrid methods have been developed (Echelmeier et al, 2019;Zhao et al, 2019;Cheng, 2020;Gru ¨nbein & Kovacs, 2019;Echelmeier et al, 2020;Ren et al, 2020;Monteiro et al, 2020;Li et al, 2018). In addition, various novel sample delivery systems, including upgrades to existing sample delivery systems, are constantly emerging for better SX practice (Gilbile et al, 2021;Martiel et al, 2021;Doppler et al, 2022;Lee et al, 2022;Patel et al, 2022;Vakili et al, 2022;Hammarstro ¨m et al, 2022). These devices can be applied to different light sources, including X-ray free-electron laser (XFELs) and synchrotron radiation (SR) sources, or both, depending on their delivery speed.…”