Abstract
High-throughput X-ray crystal structures of protein-ligand complexes are critical to pharmaceutical drug development. However, cryocooling of crystals and X-ray radiation damage may distort the observed ligand binding. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) can produce radiation-damage-free room-temperature structures. Ligand-binding studies using SFX have received only modest attention, partly owing to limited beamtime availability and the large quantity of sample that is required per structure determination. Here, a high-throughput approach to determine room-temperature damage-free structures with excellent sample and time efficiency is demonstrated, allowing complexes to be characterized rapidly and without prohibitive sample requirements. This yields high-quality difference density maps allowing unambiguous ligand placement. Crucially, it is demonstrated that ligands similar in size or smaller than those used in fragment-based drug design may be clearly identified in data sets obtained from <1000 diffraction images. This efficiency in both sample and XFEL beamtime opens the door to true high-throughput screening of protein-ligand complexes using SFX.
Highlights
The accurate determination of the structures of protein–ligand complexes is essential for drug discovery, enzymology and biotechnology
We explore the potential of this approach for rapid SFX screening of ligands/drug candidates, examining the minimum number of merged diffraction patterns required to reliably detect ligand binding and the future potential of this approach at current and planned X-ray free-electron lasers (XFELs) beamlines
Crystals grew in 1–2 days to approximate dimensions of 20– 30 mm and were transported to SPring-8 Angstrom Free Electron Laser (SACLA) at ambient temperature
Summary
The accurate determination of the structures of protein–ligand complexes is essential for drug discovery, enzymology and biotechnology. For important classes of proteins, the binding of ligands may be affected by X-ray-driven changes either in the oxidation state of redox centres within the protein or to amino-acid side chains involved in protein–ligand interactions In these cases, there is a premium on structure determination using low-dose methods. There is a premium on structure determination using low-dose methods Prime examples of this are heme enzymes, where the iron centre in resting iron(III) and high-valent iron(IV) states is exquisitely prone to reduction by solvated photoelectrons generated by the interaction of synchrotron X-rays with solvent in the crystal (see, for example, Beitlich et al, 2007; Kekilli et al, 2017).
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