Radiation damage to biological crystals is a major challenge in the determination of the crystal structure of macromolecules at conventional x-ray energies. At low energies the dominance of the photoelectric effect leads to energy deposition that causes premature radiation-induced death of the crystals. Typical sample lifetimes can be extended somewhat with the use of cryogenic temperatures, but it has been shown that it will be insufficient for third-generation, synchrotron-based beamlines. The purpose of the experiments pursued on X17B at the NSLS and ID15B was to exploit the use of high-energy x-rays in the 55keV range for the crystal structure determination of macromolecules. The data collected on a chicken lysozyme sample co-crystallized with holmium allowed explore a large variety of phasing methods: multiple-wavelength anomalous diffraction (MAD), single-wavelength anomalous diffraction (SAD) and single isomorphous replacement with anomalous scattering (SIRAS). The Bijvoet ratio, calculated with the refined occupancies is 2.5%, indicating the potential use for holmium as one of the standard heavy atoms for high energy measurements. The quality of the phases after density improvement (Figure 37) allowed automatically build the totality of the molecule for all the phasing methods tested.
Experimental electron density maps after density improvement (before model building and refinement). All 3 maps are contoured at 1σ level and calculated around the same region.
Additional data was collected at ID15B corresponding to a total cumulated exposure of 93min and an estimated cumulated dose of 1.7x106J/kg. That data was compared to the data collected on NSLS beamline X6A at 12keV with total cumulated exposure 93min and estimated dose 7x106J/kg. Crystals were grown under the same conditions with similar sizes presenting the same diffracting resolution, 1.3Å. Crystals exposed to 12keV showed the onset of radiation damage after a cumulated exposure of 18min, while no radiation damage was observed in the data collected at high energy x-ray for the total cumulated exposure. In conclusion, high-energy can be efficiently used to determine a crystal structure while minimizing radiation damage effects.
J. Jakoncic, M. Di Michiel, Z. Zhong, V. Honkimäki, Y. Jouanneau, and V. Stojanoff: Anomalous diffraction at ultra-high energy for protein crystallography, Journal of Applied Crystallography, 39, 831-841 (2006).