Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

Abstract

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.

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

“…Here, we report kHz droplet-on-demand serial crystallography, yielding rates of indexed crystals per second comparable with published MHz GDVN experiments, 27,[37][38][39][40] with a two order of magnitude reduction in sample consumption. The data were collected at the Femtosecond X-ray Experiments (FXE) beamline of the EuXFEL, marking the first report of serial protein crystallographic measurements at this beamline.…”

“…This substantial increase in the hit rate, in comparison with GDVN, enables a higher effective data rate, or number of indexed patterns per second, surpassing all but one of the previous lysozyme MHz SFX experiments conducted at the EuXFEL, shown in Table II. [37][38][39][40]47…”

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

“…At the SPB/SFX instrument, two interaction regions exist: upstream and downstream. The latter is dedicated to experiments that operate at low repetition rates and for samples that require high-viscosity extruder devices (HVEs) to jet, such as membrane protein microcrystals embedded in the viscous lipid cubic phase. , 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- m Emerald fluorescent protein microcrystals . It is possible to use mixing-HVE devices with various viscous materials, such as grease or Vaseline, − agarose, hydroxyethyl cellulose, and lards, which can cover a wide range of different metalloenzymes and membrane proteins without compromising their diffraction quality.…”

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

“…However, Holmes et al. ( 8 ) recently reported multi-hit SFX from a single lysozyme crystal using the MHz pulse structure of the European XFEL (EuXFEL). About a decade ago, Chapman et al.…”