Abstract
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
Highlights
The sulfur single-wavelength anomalous diffraction (S-SAD) phasing method allows the determination of native protein structures without requiring chemical modification or the knowledge of a homologous structure
In a previous paper (Galli et al, 2015), simulations of serial femtosecond crystallography (SFX) experiments showed that the available X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS) should provide enough photon flux to saturate sulfur ionisation at a photon energy of 6 keV, and that by reducing the highest accessible flux by about two orders of magnitude it should be possible to utilize a high fluence (HF) version of the conventional radiation-induced phasing (RIP) (Ravelli et al, 2003) workflow to obtain good quality phases of a native protein system
The native protein sample employed for this experiment was Cathepsin B (CatB) — an enzyme belonging to the class of cysteine proteases which degrade polypeptides
Summary
The sulfur single-wavelength anomalous diffraction (S-SAD) phasing method allows the determination of native protein structures without requiring chemical modification or the knowledge of a homologous structure This de novo phasing technique presents its own problems arising from the long 5.02 Awavelength at which the sulfur K-edge lies at 2.47 keV photon energy. In the case of SAD, the theory presented in the paper of Son et al predicts a reduction of the out-of-phase component of the atomic form factor proportional to the incident X-ray intensity, which would complicate anomalous phasing methods (Barends et al, 2014) This theory predicts a large amount of ionization of the heavy atoms at high intensities, which would increase the difference in the scattering strengths of these atoms for different fluences. A high intensity XFEL experiment on the actual native protein that was used for the reported simulations is described, allowing for a comparison between theory and experiment
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
