Progress in research into radiation damage in cryo-cooled macromolecular crystals

The problem of radiation damage to cryo-cooled macromolecular crystals in intense synchrotron beams has entered the mainstream of macromolecular crystallography (MX) during the last few years.A growing body of researchers worldwide are now carrying out systematic investigations into different aspects of this problem.The Fourth International Workshop on X-ray Radiation Damage to Biological Crystalline Samples was held on 7-8 March 2006 at SPring-8 in Japan and was attended by 80 researchers, the meeting being generously funded by SPring-8, Hyogo, Japan, and the Photon Factory in Tsukuba, Japan.The series of workshops on X-ray Damage to Biological Crystalline Samples aims to discuss and disseminate the latest research, often prior to publication, on the understanding, control, correction and possible use of radiation damage occurring during macromolecular crystallography experiments.Papers from the second (2001) and third (2003) workshops can be found in special issues of the Journal of Synchrotron Radiation (RD2, 2002; RD3, 2005).In this issue of the Journal of Synchrotron Radiation, there are 12 papers covering various aspects of current research into this area, including nine arising from the presentations made during the workshop.It is now generally recognized that information deduced from three-dimensional biological structures can be compromised because of radiation-induced effects.In addition to the general degradation of diffraction properties associated with increasing dose, specific structural damage is observed to occur at 100 K in a well defined sequence, with the disulfide bond becoming disordered by breakage and delocalization, followed by the decarboxylation of aspartates and glutamates (Weik et al., 2000;Burmeister, 2000; Ravelli & McSweeney, 2000).Metal binding proteins are partly reduced during X-ray exposure (Berglund et al., 2002;Yano et al., 2005), and active sites seem particularly susceptible to damage (Weik et al., 2000).These observations imply that an awareness of radiation damage effects is essential before any mechanistic conclusions are drawn from, for instance, the oxidation state of a metal atom in an active site.Increasingly, the processes of radiation damage are being used to enhance phase information and thereby provide more accurate starting maps.As the trend towards multidisciplinary biological research continues, it becomes important to consider how experience from other scientific fields, for instance spectroscopic tools and electron microscopy, may aid research in MX.Systematic experiments on the characteristics of radiation damage over the last five years have resulted in the identification of a number of the important beam and crystal parameters governing its rate of progression and character (reviewed by Ravelli & Garman, 2006).Research reports in this Journal of Synchrotron Radiation issue can be loosely grouped into five different areas: investigations into the general dependencies of radiation damage on various parameters, the effects of damage on phasing power and ways to correct for it, the use of optical and Raman spectroscopy to study aspects of damage, the study of heating effects induced by the beam in cryo-cooled crystals, and, lastly, experience in electron microscopy and how this might be applied to MX.In this issue, Shimizu et al. (2007) report the analysis of various radiation damage indicators in lysozyme crystals as a function of incident photon energy, concluding that between 6.5 keV and 33 keV there is no observable energy dependence of radiation damage on dose.The possible correlation between the rate of radiation damage and the solvent accessibility of glutamates and aspartate residues in the halophilic enzyme malate dehydrogenase is explored by Fioravanti et al. (2007), who find there to be no such correlation.Borek et al. (2007) describe a range of diffraction experiments carried out on crystals of several different proteins to investigate the effects of cryogen temperature on various processing statistics and on the observed specific structural damage.