Abstract
Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage. However, de novo structure determination, i.e., without prior structural knowledge, is complicated by the inherent inaccuracy of serial femtosecond crystallography data. By its very nature, serial femtosecond crystallography data collection entails shot-to-shot fluctuations in X-ray wavelength and intensity as well as variations in crystal size and quality that must be averaged out. Hence, to obtain accurate diffraction intensities for de novo phasing, large numbers of diffraction patterns are required, and, concomitantly large volumes of sample and long X-ray free-electron laser beamtimes. Here we show that serial femtosecond crystallography data collected using simultaneous two-colour X-ray free-electron laser pulses can be used for multiple wavelength anomalous dispersion phasing. The phase angle determination is significantly more accurate than for single-colour phasing. We anticipate that two-colour multiple wavelength anomalous dispersion phasing will enhance structure determination of difficult-to-phase proteins at X-ray free-electron lasers.
Highlights
Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage
Two-colour data collection was performed at beamline 3 (BL3) in the DAPHNIS chamber[31] using a multiport charge coupled device (MPCCD) detector[32]
SPring-8 Angstrom Compact free-electron LAser (SACLA) operated at 30 Hz and simultaneously delivered two-colour X-ray pulses of 10 fs duration and nominally 7.0 keV (λ = 1.770 Å) and 9.0 keV (λ = 1.378 Å) photon energy of 0.14 mJ average power
Summary
Serial femtosecond crystallography at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems prone to radiation damage. We show that serial femtosecond crystallography data collected using simultaneous two-colour X-ray free-electron laser pulses can be used for multiple wavelength anomalous dispersion phasing. We anticipate that two-colour multiple wavelength anomalous dispersion phasing will enhance structure determination of difficult-to-phase proteins at X-ray free-electron lasers. A great deal of data must be collected; the multiplicity of measurements for a given reflection being typically several 100- to 1,000-fold depending on the phasing method and signal strength This demands significant quantities of sample and of XFEL beam time, both of which are typically precious and often limiting. The recently established two-colour operation of the SPring-8 Angstrom Compact free-electron LAser (SACLA) in Japan[26] opened up a novel possibility of collecting two SFX datasets simultaneously, without doubling the amount of sample used
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