X-rays intended to elucidate the structure of biomolecules may actually damage and alter key parts of the molecules. A research team led by a group from Lawrence Berkeley National Laboratory (in collaboration with researchers from Max-Planck-Institut Mülheim, ESRF, SSRL, and TU Berlin and Freie Universität, Berlin) discovered this while investigating the Mn₄Ca complex, a site crucial for splitting water into oxygen during photosynthesis. They were using x-ray absorption spectroscopy to learn about the Mn₄Ca structure in crystals of the protein known as photosystem II. They found that the metallo-protein active site, where the water splitting takes place in the protein, had been damaged at certain x-ray doses. In fact, the site had completely changed its structure. This demonstrated that the structural information deduced by using x-ray diffraction is not reliable.

The research suggests that the x-ray dose that can cause such damage to the active site in metallo-proteins—a typical dose for diffraction studies—may be lower than previously believed. However, the new investigation showed that lowering the temperature of the sample and using higher energy x-rays can dramatically reduce damage, thus providing a way for working around the problem.