Abstract
Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.
Highlights
X-ray free-electron lasers (XFELs) generate very short/ intense pulses that enable the collection of diffraction data before the destruction of the specimen (Neutze et al, 2000)
The crystals were back-soaked for 48 h and soaked for 24 h at room temperature using the same solutions as used for the microcrystals apart from a reduced ZA concentration of 30 mM in the soaking solution
Three SFX and two synchrotron radiation (SR) structures of thermolysin have been determined at comparable resolutions in the range 1.9–2.3 A (Table 1 and Supplementary Fig. S1)
Summary
X-ray free-electron lasers (XFELs) generate very short/ intense pulses that enable the collection of diffraction data before the destruction of the specimen (Neutze et al, 2000). This ‘diffraction-before-destruction’ principle of XFELs has successfully been applied in serial femtosecond crystallography (SFX), in which hundreds of thousands of single-shot diffraction images from randomly oriented microcrystals at room temperature are merged to determine a crystal structure (Chapman et al, 2011; Boutet et al, 2012). A damage-free structure from SFX could account for the proton-transfer mechanism of nitrite reductase (Fukuda et al, 2016).
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