Abstract
The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.
Highlights
Since the initial proof-of-principle serial crystallography experiments[1,2] performed in 2009 at the first hard X-ray free-electron laser (XFEL), the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, XFEL technologies have been further developed that include novel techniques to improve the growth of nano-/microcrystals, deliver samples, and analyze SFX data
The term MHz crystallography was first introduced in the reporting of SFX at the European XFEL (EuXFEL) with concanavalin A, concanavalin B, and lysozyme by Grünbein et al.[10] and on lysozyme and β-lactamase by Wiedorn et al.[11], where MHz refers to the repetition rate within a pulse train
In SFX, the sample is delivered in a serial way to the beam, commonly in a jet of crystals in their mother liquor[2]. It must be rapidly replenished between the XFEL pulses, removing the sample destroyed by the previous pulse
Summary
Since the initial proof-of-principle serial crystallography experiments[1,2] performed in 2009 at the first hard XFEL, the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory, XFEL technologies have been further developed that include novel techniques to improve the growth of nano-/microcrystals, deliver samples, and analyze SFX data. In run 2, the rate was increased to 120 pulses per train, delivering 1200 pulses per second, and correspondingly, at least a tenfold increase in the data collection rate compared with other hard XFELs. The term MHz crystallography was first introduced in the reporting of SFX at the EuXFEL with concanavalin A, concanavalin B, and lysozyme by Grünbein et al.[10] and on lysozyme and β-lactamase by Wiedorn et al.[11], where MHz refers to the repetition rate within a pulse train. Current sample injection technology results in substantial sample wastage between pulse trains[11] This poses a challenge for diminishing sample consumption, despite the MHz repetition rate within a pulse train. The fastest Xray detector is the newly developed Adaptive Gain Integrating Pixel Detector (AGIPD), which can collect up to 352 images per train (potentially storing 3520 images per second)[15,16]
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