Effect of X-ray free-electron laser-induced shockwaves on haemoglobin microcrystals delivered in a liquid jet

Abstract

X-ray free-electron lasers (XFELs) enable obtaining novel insights in structural biology. The recently available MHz repetition rate XFELs allow full data sets to be collected in shorter time and can also decrease sample consumption. However, the microsecond spacing of MHz XFEL pulses raises new challenges, including possible sample damage induced by shock waves that are launched by preceding pulses in the sample-carrying jet. We explored this matter with an X-ray-pump/X-ray-probe experiment employing haemoglobin microcrystals transported via a liquid jet into the XFEL beam. Diffraction data were collected using a shock-wave-free single-pulse scheme as well as the dual-pulse pump-probe scheme. The latter, relative to the former, reveals significant degradation of crystal hit rate, diffraction resolution and data quality. Crystal structures extracted from the two data sets also differ. Since our pump-probe attributes were chosen to emulate EuXFEL operation at its 4.5 MHz maximum pulse rate, this prompts concern about such data collection.

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

“…Quality dark frames (or time 0) together with various other experiments validated the structural information that was obtained [34,73]. [33]. On the left is the photocaged NO and the corresponding de-caged molecules after laser photoexcitation at 308 nm are on the right [74].…”

“…In conclusion, although photocages enable a method that can provide the fastest timescale triggering of a system (similar accessible timescales up to fs are allowed by the Figure 3. The photocage molecule used in [33]. On the left is the photocaged NO and the corresponding de-caged molecules after laser photoexcitation at 308 nm are on the right [74].…”

“…Time-resolved serial femtosecond crystallography (TR-SFX) is defined by the study of biomacromolecular reactions observed with time resolutions as rapid as femtosecond-scale using SFX [15,21,22]. Since the first published TR-SFX experiment in 2012 at LCLS [23], many studies have been reported [24][25][26][27][28][29][30][31], including those conducted at the European XFEL that made use of its ultra-fast repetition rate [18,32,33]. As time-resolved studies require much more data to be collected in comparison to regular SFX (N times more, where N is the number of desired time points to obtain reliable difference electron density maps), the megahertz repetition rate represents a significant advantage in data collection time [14].…”

3D printed devices and infrastructure for liquid sample delivery at the European XFEL

“…Another important constraint is the enormous energy deposited in a single ultrashort X-ray pulse (ca 2.5 mJ at SASE1), which vaporizes a segment of the jet, leading to gaps in the liquid column. Each explosion further generates a shock wave that propagates both upstream and downstream (Stan et al, 2016;Gru ¨nbein et al, 2021). The spacing between two gaps is usually of the order of several tens of micrometres (Chavas et al, 2015) and roughly describes the displacement between two intra-train pulses, which allows the determination of jet velocities (Gru ¨nbein, Wiedorn, Oberthu ¨r et al, 2018).…”

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

“…Each train contained 202 X-ray pulses with approximately 40 fs fullwidth at half-maximum (FWHM) pulse duration and about 1.5 mJ pulse energy. The pulse-repetition rate within a pulse train was 564 kHz, a reduction from the possible 4.5 MHz to avoid pristine, upstream jet volumes being affected by previous X-ray pulses (Yefanov et al, 2019;Pandey et al, 2020;Gru ¨nbein et al, 2021). Given a flow rate of about 80 ml min À1 (Table 2) and an assumed jet diameter of 8 mm, the jet advances 26.5 m in a second.…”