Membrane protein megahertz crystallography at the European XFEL

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.

Fig. 1

Crystals of PSI. a Variable-size PSI crystal distribution, grown by ultrafiltration. b DLS (ten 18-s scans, numbered consecutively from 1 to 10, “Scan Number”) of nanocrystals with uniform size distribution used for seeding. c PSI crystals of uniform size of 5 × 5 × 15 µm grown by using the RAMS method

Fig. 2

Unit cell distribution, diffraction pattern, and indexing rates from PSI MHz SFX. a Unit cell distributions of PSI microcrystals showing narrowly distributed monoclinic unit cells. The red line shows a Gaussian function fit to the unit cell constant distribution, and the corresponding peak value is listed in each subpanel. b Representative X-ray diffraction pattern with pixels in high-gain mode shown in black, and medium- or low-gain mode shown in red. Resolution rings are shown and labeled accordingly. c The number of hits (red) and indexed patterns (blue, ~93% of hits) for each pulse

Fig. 3

Electron density map (2Fo–Fc at 1.5σ) and model of various PSI structural elements of the XFEL structure of PSI. In all images, protein is colored cyan, chlorophyll (Chl) molecules are colored green, β-carotenes are colored orange, and lipids are colored yellow. In panels b–e, nitrogen atoms are colored blue, oxygen atoms are colored red, and magnesium atoms are colored bright green. a A slice through the center of electron density of a monomer of PSI is shown, b the electron density of the “special pair” of Chls, P₇₀₀, c a β-carotene molecule, d the 4Fe–4S cluster, F_X, and e the phosphatidylglycerol lipid headgroup axial coordination of a Chl molecule

Fig. 4

Comparison of SFX structures from the EuXFEL. The structure of the trimeric PSI determined in this study is shown to scale with the four protein structures previously solved at the EuXFEL by using MHz repetition rates for comparison. Views from the membrane plane (top) and membrane normal (bottom) are shown for the PSI trimer, with major and minor axes denoted. Protein subunits are colored individually