Time-Resolved Serial Femtosecond Crystallography at the European XFEL

The European XFEL (EuXFEL) is an X-ray source that produces femtosecond X-ray pulses at megahertz repetition rates. Time-resolved crystallographic investigations on biological reactions constitute an important class of experiments. We demonstrate how such a reaction is followed at the EuXFEL.
Free electron lasers for hard X-rays (XFELs) provide intense femtosecond quasi-monochromatic X-ray pulses. Since, as the name suggests, the XFEL is a laser, the X-ray beam can be focused without losses to focal spots with small cross sections. Due to this, the number of photons per unit area and 0.1% bandwidth in a single X-ray pulse can be more than six orders of magnitude larger than that at other modern X-ray sources. This makes it possible to produce a diffraction pattern from tiny micrometer and sub-micrometer sized protein crystals. After the X-ray pulse the crystal disintegrates due to radiation damage. Since disintegration is slower than diffraction, the diffraction pattern originates from a crystal that is essentially free of radiation damage. This has been named “the diffraction before destruction principle”. That this principle works at a free electron laser (FEL) has been shown in 2006 by Henry Chapman and colleagues using a cowboy etched in a silicone nitride foil [1]. After one pulse of ultraviolet (UV) radiation from a FEL called FLASH located at the Deutsche Elektronen-SYnchrotron (DESY) in Hamburg, the etching was destroyed, yet the image of the figure could be reconstructed from the collected diffraction pattern. Interestingly, already in the 1950s this principle has been demonstrated on a much grander scale using the fastest film camera at this time and a thermonuclear explosive device mounted on a steel tower. A few microseconds after ignition, a perfect image of the intact tower is obtained, yet the tower is completely destroyed afterwards by the might of the nuclear explosion. Here, instead of probing cowboys etched in silicone with UV radiation, or towers illuminated by a nuclear explosion, tiny macromolecular crystals are interrogated by hard X-rays. Similar to the etching (or the tower), the microcrystals are also destroyed. Additional diffraction patterns can only be collected from new and pristine crystals delivered one by one, in a serial fashion, to the X-ray pulses. Accordingly, this method is called “serial femtosecond crystallography” (SFX). SFX revolutionized crystallography, since now, damage free structures can be obtained at room temperature. New software solutions needed to be developed (and are available e.g. from the Center of Free Electron Laser Science at DESY, Hamburg) that are capable of stitching together a crystallographic dataset of reflection intensities from millions of diffraction patterns of which the majority are blanks and do not even contain Bragg reflections.

Time-resolved serial femtosecond crystallography (TR-SFX) at the European XFEL. Microcrystals are injected by a gas dynamic virtual nozzle. A reaction is initiated by blue laser pulses, and the structure is probed during the reaction by the XFEL pulses. The diffraction-before-destruction principle ensures that essentially radiation damage free structures are observed although the crystals disintegrate (picture copyright: European XFEL/Blue Clay Studios).

When a reaction is initiated in the tiny crystals and the crystals are interrogated a time delay Δt after this by the XFEL pulse, the progress of the reaction can be probed and characterized by X-ray structures. This method is called time-resolved (TR) SFX. Displacements of atoms can be identified through shifted (altered) electron density distributions in the macromolecule. Multiple ways to initiate reactions are conceivable. It is quite straightforward to use short and intense optical laser pulses to start the reaction, and probe the reaction some time delay after. This strategy is called the “pump-probe” approach which works only if a photo-reactive cofactor is present in the biological macromolecule. The photoactive yellow protein (PYP) is such a macromolecule. It harbors a para-coumaric acid (pCA) chromophore, who undergoes a trans to cis isomerization after absorption of a photon in the blue (visible) spectral range. After completing a photocycle in about 100 – 500 ms the PYP returns back to its dark state. We recently pioneered TR-SFX at the Linac Coherent Light Source (LCLS), an XFEL located in Menlo Park, California. We reported the first difference electron density map with near atomic resolution obtained at any XFEL [2]. We then explored the trans to cis isomerization of the pCA chromophore on the femtosecond time scale, and up to 3 ps [3].  Previously, the PYP photocycle was also characterized by synchrotron based time-resolved crystallography on time scales larger than 100 ps. Hence, a gap from 3 ps to 100 ps remained uncharted. We attempted to close the gap by pioneering TR-SFX data collection at the European XFEL (EuXFEL). The EuXFEL is a high repetition rate XFEL that is a 3 km long structure originating at DESY in Hamburg, and stretches all the way to another German federal state called Schleswig-Holstein. The EuXFEL is driven by a superconducting linear accelerator. Due to this, it can produce bursts of X-ray pulses with megahertz (MHz) repetition rates. To make use of these high pulse rates, a tunable laser must be used that can produce high power, femtosecond optical laser pulses at rates that match the high-repetition X-ray pulse rates at the EuXFEL. This laser, which is quite an engineering marvel, has recently made available to users at the EuXFEL. To reliably achieve time delays on the picosecond time range required for our experiments on PYP, optical pulses from this laser must be properly synchronized with picosecond precision to the MHz X-ray pulses. This was a major achievement for the beamline staff and the laser specialists [4]. For the first time, we succeeded to collect TR-SFX data at the EuXFEL [5]. We were able to calculate difference electron (DED) maps at the EuXFEL at time-delays of 10 ps, 30 ps and 80 ps in the PYP photocycle. As a control, TR-SFX data at time delays between 0.9 μs and 5 μs were also collected. The picosecond DED maps show strong, and chemical meaningful difference features, which can be interpreted by appropriate structural models. These models then close the mentioned gap in the photocycle, and provide detailed views of a non-ergodic macromolecular relaxation (in PYP) on the ultrafast time-scale. This experiment shows that TR-SFX is possible with MHz pulse rates. Other methods to initiate reactions are conceivable. Most promising is an approach we called “mix-and-inject serial crystallography” (MISC) designed to investigate enzyme catalyzed reactions. Tiny enzyme crystals are simply mixed with substrate. The reaction is initiated by diffusion of substrate into the crystals. In microcrystals diffusion times can be sub-milliseconds, which is much faster than most enzymes’ catalytic cycles. As a result, the enzymatic turnover can be observed in real time and structurally characterized [6]. Implementing MISC at the EuXFEL must be a major goal, since enzyme catalyzed reactions can be biomedically important. The EuXFEL may become a tool that contributes to saving lives. In any case the high repetition rates available at the EuXFEL will greatly speed up data collection and will facilitate comprehensive investigations on bio-macromolecular reactions with time-resolved methods of any flavor that benefit from X-ray pulses.

1.  Chapman, H. N.; Barty, A.; Bogan, M. J.; Boutet, S.; Frank, M.; Hau-Riege, S. P.; Marchesini, S.; Woods, B. W.; Bajt, S.; Benner, H.; London, R. A.; Plonjes, E.; Kuhlmann, M.; Treusch, R.; Dusterer, S.; Tschentscher, T.; Schneider, J. R.; Spiller, E.; Moller, T.; Bostedt, C.; Hoener, M.; Shapiro, D. A.; Hodgson, K. O.; Van der Spoel, D.; Burmeister, F.; Bergh, M.; Caleman, C.; Huldt, G.; Seibert, M. M.; Maia, F. R. N. C.; Lee, R. W.; Szoke, A.; Timneanu, N.; Hajdu, J., Femtosecond diffractive imaging with a soft-X-ray free-electron laser. Nat Phys 2006, 2 (12), 839-843.

2.  Tenboer, J.; Basu, S.; Zatsepin, N.; Pande, K.; Milathianaki, D.; Frank, M.; Hunter, M.; Boutet, S.; Williams, G. J.; Koglin, J. E.; Oberthuer, D.; Heymann, M.; Kupitz, C.; Conrad, C.; Coe, J.; Roy-Chowdhury, S.; Weierstall, U.; James, D.; Wang, D.; Grant, T.; Barty, A.; Yefanov, O.; Scales, J.; Gati, C.; Seuring, C.; Srajer, V.; Henning, R.; Schwander, P.; Fromme, R.; Ourmazd, A.; Moffat, K.; Van Thor, J. J.; Spence, J. C.; Fromme, P.; Chapman, H. N.; Schmidt, M., Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein. Science 2014, 346 (6214), 1242-6.

3.  Pande, K.; Hutchison, C. D. M.; Groenhof, G.; Aquila, A.; Robinson, J. S.; Tenboer, J.; Basu, S.; Boutet, S.; Deponte, D.; Liang, M.; White, T.; Zatsepin, N.; Yefanov, O.; Morozov, D.; Oberthuer, D.; Gati, C.; Subramanian, G.; James, D.; Zhao, Y.; Koralek, J.; Brayshaw, J.; Kupitz, C.; Conrad, C.; Roy-Chowdhury, S.; Coe, J. D.; Metz, M.; Lourdu Xavier, P.; Grant, T. D.; Koglin, J.; G., K.; Fromme, R.; Srajer, V.; Henning, R.; Spence, J. H. C.; Ourmazd, A.; Schwander, P.; Weierstall, U.; Frank, M.; Fromme, P.; Barty, A.; Chapman, H. N.; Moffat, K.; Van Thor, J. J.; Schmidt, M., Femtosecond Structural Dynamics Drives the Trans/Cis Isomerization in Photoactive Yellow Protein. Science 2016, 352 (6286), 725-729.

4.  Kirkwood, H. J.; Letrun, R.; Tanikawa, T.; Liu, J.; Nakatsutsumi, M.; Emons, M.; Jezynski, T.; Palmer, G.; Lederer, M.; Bean, R.; Buck, J.; Cafisio, S. D.; Graceffa, R.; Grunert, J.; Gode, S.; Hoppner, H.; Kim, Y.; Konopkova, Z.; Mills, G.; Makita, M.; Pelka, A.; Preston, T. R.; Sikorski, M.; Takem, C. M. S.; Giewekemeyer, K.; Chollet, M.; Vagovic, P.; Chapman, H. N.; Mancuso, A. P.; Sato, T., Initial observations of the femtosecond timing jitter at the European XFEL. Optics letters 2019, 44 (7), 1650-1653.

5.  Pandey, S.; Bean, R.; Sato, T.; Poudyal, I.; Bielecki, J.; Cruz Villareal, J.; Yefanov, O.; Mariani, V.; White, T. A.; Kupitz, C.; Hunter, M.; Abdellatif, M.; Bajt, S.; Bondar, V.; Echelmeier, A.; Doppler, D.; Emons, M.; Frank, M.; Fromme, R.; Gevorkov, Y.; Giovanetti, G.; Jiang, M.; Kim, D.; Kim, Y.; Kirkwood, H.; Klimovskaia, A.; Knoska, J.; Koua, F. H. M.; Letrun, R.; Lisova, S.; Maia, L.; Mazalova, V.; Meza, D.; Michelat, T.; Ourmazd, A.; Palmer, G.; Ramilli, M.; Schubert, R.; Schwander, P.; Silenzi, A.; Sztuk-Dambietz, J.; Tolstikova, A.; Chapman, H. N.; Ros, A.; Barty, A.; Fromme, P.; Mancuso, A. P.; Schmidt, M., Time-Resolved Serial Femtosecond Crystallography at the European XFEL. Nature methods 2019, https://doi.org/10.1038/s41592-019-0628-z.

6.  Olmos, J. L., Jr.; Pandey, S.; Martin-Garcia, J. M.; Calvey, G.; Katz, A.; Knoska, J.; Kupitz, C.; Hunter, M. S.; Liang, M.; Oberthuer, D.; Yefanov, O.; Wiedorn, M.; Heyman, M.; Holl, M.; Pande, K.; Barty, A.; Miller, M. D.; Stern, S.; Roy-Chowdhury, S.; Coe, J.; Nagaratnam, N.; Zook, J.; Verburgt, J.; Norwood, T.; Poudyal, I.; Xu, D.; Koglin, J.; Seaberg, M. H.; Zhao, Y.; Bajt, S.; Grant, T.; Mariani, V.; Nelson, G.; Subramanian, G.; Bae, E.; Fromme, R.; Fung, R.; Schwander, P.; Frank, M.; White, T. A.; Weierstall, U.; Zatsepin, N.; Spence, J.; Fromme, P.; Chapman, H. N.; Pollack, L.; Tremblay, L.; Ourmazd, A.; Phillips, G. N., Jr.; Schmidt, M., Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography. BMC Biol 2018, 16 (1), 59.

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