Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers

Abstract

X-ray crystallography at X-ray free-electron laser (XFEL) sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy (XES), both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing new insights into the interplay between the protein structure/dynamics and chemistry at an active site. Implementing such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly impacts the data quality. We present here a new, robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

X-ray Emission Spectroscopy as an in Situ Diagnostic Tool for X-ray Crystallography of Metalloproteins Using an X-ray Free-Electron Laser

“…The details of the sample preparation, experimental set-up, and data analysis, as well as other relevant details, are in the Supporting Information. 13, 26 The set-up used a von Hamos spectrometer 9, 27 to measure the entire Kβ 1,3 emission spectrum on a shot-by-shot basis. 9, 16, 17, 27 The signal was collected on an ePix100 detector 28 with a pixel size of 50 μm.…”

“…10-13 This method also allows us to confirm the integrity of the sample and the catalytic turnover, by providing an in situ diagnostic tool for determining the oxidation state of the metal in the active sites. The use of XES is well suited because the excitation energy can be the same as for XRD, therefore enabling simultaneous data collection.…”

“…The XRD/XES data collection setup, which can be used with either a liquid microjet injector 20, 21 or the more recent Drop-on-Tape (DOT) 13 method is shown in Fig. 1.…”

Perspective: Opportunities for ultrafast science at SwissFEL

“…This made it necessary to develop new ways of collecting data, commonly termed as serial femtosecond crystallography (SFX) because a large number of crystals (often thousands) are exposed in a serial fashion to the femtosecond XFEL pulses. Methods to deliver crystals include the fixed target approach, where crystals are scanned on chip-like devices, 109,221 or the drop-on demand method relying on acoustic droplet ejection coupled with a conveyor belt drive 222 . To date, the most successful and prominent method is based on a continuous flow of nano- to micrometer sized crystals injected with a jet device 87,223 .…”

Analysis of XFEL serial diffraction data from individual crystalline fibrils

“…Such crystals are easier to obtain than those of at least tens of micrometres across that are required by synchrotron sources. Crystals are usually injected into the XFEL pulse path through a nozzle as a suspension in a liquid jet Nelson et al, 2016;Oberthuer et al, 2017) oriented perpendicular to the incident X-ray path, although other sample-delivery methods are also in use Chavas et al, 2015;Sierra et al, 2015;Roedig et al, 2016;Martin-Garcia et al, 2016;Fuller et al, 2017). The maximum pulse repetition rate at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory is 120 Hz, and diffraction patterns are usually recorded at this rate (Bostedt et al, 2016).…”