Megahertz serial crystallography

Abstract

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Fig. 1

Megahertz serial crystallography. Pulses from the European XFEL were focused on the interaction region using a set of Beryllium lenses. Protein crystals in crystallization solution were introduced into the focused XFEL beam using a liquid jet of 1.8 µm diameter moving at speeds between 50 m/s and 100 m/s. Diffraction from the sample was measured using an AGIPD, which is capable of measuring up to 3520 pulses per second at megahertz frame rates. In-situ jet imaging (inset) showed that the liquid column does explode under the X-ray illumination conditions of this experiment using a jet with a speed of 100 m/s, but that the liquid jet recovered in less than 1 μs to deliver fresh sample in time for arrival of the next X-ray pulse. Images and movies of jets at different speeds are included in the supplementary material

Fig. 2

Diffraction pattern from HEWL. Diffraction pattern from a single HEWL microcrystal measured using MHz pulses of 50 fs duration X-rays at 9.3 keV using the AGIPD 1M detector in the SPB/SFX instrument. Dynamic gain switching of the AGIPD detector enables simultaneous low noise and high dynamic range: each pixel has three gain settings which are automatically selected depending on the per-pixel cumulative intensity to simultaneously maximize sensitivity and dynamic range. Image clipped at 2600 counts to show content, full dynamic range of brightest spots extends to 109,000 counts

Fig. 3

Images of interaction of the EuXFEL liquid jet for the first 5 pulses in a train. Jets in the range of 50–100 m/s recover in time for the next pulse (first three rows), whereas slower jets of the type commonly used at LCLS do not recover in time for the next XFEL pulse at MHz repetition rates (bottom row). The bottom line provides linkage back to the results presented in ref. . Red line shows the intersection point with X-ray pulses. Images obtained by synchronized laser back illumination. Movies with finer time steps are included as supplementary material

Fig. 4

HEWL diffraction was measured on all pulses in the pulse train. a Hit fraction as a function of pulse number indicates that crystals are hit randomly on any pulse within the MHz EuXFEL pulse train, and not only on the first pulse in the pulse train. b Indexable lattices were equally distributed among the MHz XFEL pulse trains and no sign of degradation in data quality is observed through the pulse train as measured by the overall CC* for subsets of the data corresponding to each pulse. c CrystFEL resolution estimate as a function of X-ray pulse within a train shows no decrease in estimated resolution through the course of the pulse train. d CC* for data separated from each pulse indicates similar data quality for each pulse in the pulse train. Merging all pulses produces higher data quality (as expected). e Correlation of merged data from the first pulse relative to each subsequent pulse in the pulse train indicates that data are similar on each pulse to the limit of data quality available in this experiment. Both d and e are generated from the same stream files used for structure determination sorted according to pulse ID

Fig. 5

Electron density map for HEWL by MHz SFX. a 2Fo-Fc map at 1 sigma overlaid on Fo-Fc map at 3 sigma from molecular replacement using a solvent-free version of the 4ET8 lysozyme structure as the starting model. b Integrity of the measured data is verified by complete rebuilding of the structure from a truncated starting model after removal of residues 1–16 and 40–60 of the polypeptide chain using Autobuild

Fig. 6

Structure of CTX-M-14 β-lactamase determined by MHz SFX. a 2Fo-Fc map at 1 sigma overlaid on Fo-Fc map at 3 sigma around covalently bound avibactam from molecular replacement using a solvent-free version of the 5TWD β-lactamase structure from ref. as the starting model. b Representation of covalently bound avibactam to OG of Ser70, stabilized by hydrogen bonds and hydrophobic interactions with surrounding amino acids of CTX-M-14. Figure was prepared using Ligplot