Abstract
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.
Highlights
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unique opportunities for structural biologists (Chapman et al, 2011; Martin-Garcia et al, 2016; Orville, 2020)
Several SFX studies have demonstrated that de novo structure determination from SFX data is possible using well characterized and strongly diffracting model systems via singlewavelength anomalous diffraction (SAD) (Barends et al, 2014; Hunter et al, 2016; Nakane et al, 2016; Sugahara et al, 2017; Yamashita et al, 2017), native SAD (Batyuk et al, 2016; Nakane et al, 2015; Nass et al, 2016), isomorphous replacement (Yamashita et al, 2015), and novel phasing methods such as simultaneous multi-wavelength anomalous diffraction (MAD) with two-color X-ray pulses (Gorel et al, 2017) and high-intensity MAD (Son et al, 2011), but to date only one previously unknown structure of BinAB was determined at an XFEL (Colletier et al, 2016)
To test whether large-BW XFEL pulses offer advantages for SFX when compared with pulses with smaller typical spontaneous emission (SASE) XFEL BW postulated earlier (Dejoie et al, 2013; White et al, 2013), we used well diffracting thaumatin microcrystals
Summary
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unique opportunities for structural biologists (Chapman et al, 2011; Martin-Garcia et al, 2016; Orville, 2020). Several SFX studies have demonstrated that de novo structure determination from SFX data is possible using well characterized and strongly diffracting model systems via singlewavelength anomalous diffraction (SAD) (Barends et al, 2014; Hunter et al, 2016; Nakane et al, 2016; Sugahara et al, 2017; Yamashita et al, 2017), native SAD (Batyuk et al, 2016; Nakane et al, 2015; Nass et al, 2016), isomorphous replacement (Yamashita et al, 2015), and novel phasing methods such as simultaneous multi-wavelength anomalous diffraction (MAD) with two-color X-ray pulses (Gorel et al, 2017) and high-intensity MAD (Son et al, 2011), but to date only one previously unknown structure of BinAB was determined at an XFEL (Colletier et al, 2016)
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