Radiation damage in serial femtosecond crystallography studied in hemoglobin.

A central question in macromolecular crystallography is what X-ray free-electron laser pulse duration is required to obtain damage-free structural information. We compare serial femtosecond crystallography data from hemoglobin crystals using 3 and 10 fs pulses at comparable peak intensities (∼3 × 1017 W cm-2) and fixed photon energy (7.15 keV). Structural refinement produced very similar models and resolution-dependent data-quality indicators remained comparable. Both datasets also retained sufficient anomalous signal for phasing. These observations support the conclusion that high-resolution scattering was preserved under both pulse durations. Hybrid collisional-radiative and molecular-dynamics simulations show that under typical experimental conditions, atomic form-factor changes are negligible (<1%) and atomic displacements fall below the resolution limit imposed by the X-ray photon energy and the geometry of the detector. The combined experimental and theoretical results indicate that 10 fs pulses are adequate to obtain damage-free protein structures using femtosecond crystallography under the intensity conditions explored.