The goal of the high-risk/high-gain proposal ?Radioprotectants for structural biology? is to develop additives to slow down the progress of radiation damage in X-ray crystallography and cryo-EM single particle reconstruction (cryo-EM SPR). Both methods are of immense significance to the NIH mission, because the structural models that they generate are used to study and to modulate cellular functions in health and disease. However, X-ray crystallography and cryo-EM are affected by detrimental chemical reactions induced by the ionizing radiation that is used in both fields to generate structural information. The radiation-induced processes ultimately destroy the sample, but even before the destruction, the chemical and physical integrity of the sample becomes compromised, and consequently, radiation-damaged data become harder to interpret or sometimes cannot be interpreted at all. The decay of the measured signals in both fields is described by a resolution-dependent factor (the scaling B-factor) which can be calculated very accurately in X-ray crystallography and is also used in cryo-EM SPR. We propose to test with rigorous methods various additives targeting specific radiation-induced reactions so that the lifespan of samples used in X-ray crystallography and cryo-EM can be extended. We will analyze how select additives affect the scaling B-factor for a group of benchmark proteins that can be used both in X-ray crystallography and in cryo-EM experiments. We will also computationally decompose radiation damage into the overall radiation damage described by the B-factor and other components of radiation-induced changes in the measured data. These components will be analyzed for their sources, quantified, and validated in multi- temperature experiments and experiments involving various rates of data collection so that the impact of dose- rate effects can be determined. In parallel, we will perform cryo-EM experiments with the same compounds, to analyze whether the structure of amorphous ice in the sample and beam-induced motion of the sample are affected by these additives and if so, to what extent. We will also determine the capacity of each substance for scavenging. The technology that we plan to develop will expand the range of applicability of both techniques. In cryo-EM, longer exposures will make it possible to study molecules with proportionally lower mass and will help to correct currently uncorrectable components of beam-induced motion. In X-ray crystallography, larger doses will result in full or at least more complete data sets from smaller crystals and will permit more reliable recovery of the state without radiation damage?the zero-dose extrapolated state?for macromolecules containing highly reactive redox systems.
X-ray crystallography and cryo-EM single particle reconstruction (cryo-EM SPR) are two major methods of structural biology which provide information about the spatial organization of macromolecules and their complexes, with atomic resolution. This proposal aims to dramatically extend the applicability of these two techniques by addressing the problem of radiation damage which is introduced into samples during X-ray and cryo-EM experiments by the ionizing radiation used by these two methods to generate data. The proposed approach is highly relevant to the NIH mission because, if successful, it will advance thousands of structural projects that use crystallography and cryo-EM SPR to design better treatments and to understand cellular processes in health and disease.
