X-ray tomographic reconstruction of macromolecular samples

Abstract

The anomalous scattering properties of innate sulfur for proteins and phosphorus for DNA and RNA can be used to solve the phase problem in macromolecular crystallography (MX)viathe single-wavelength anomalous dispersion method (SAD). However, this method, which is carried out at longer X-ray wavelengths (1.5–2.5 Å), is still not a routine tool in MX. The increased absorption from both air and sample associated with the use of longer X-ray wavelengths presents a key difficulty. The absorption can be corrected for through empirical algorithms, provided truly redundant data are available. Unfortunately, weakly diffracting macromolecular crystals suffer from radiation damage, resulting in a dose-dependent non-isomorphism which violates the assumption upon which these empirical algorithms are based. In this report, X-ray microtomography is used to reconstruct the three-dimensional shapes of vitrified macromolecular crystals including the surrounding solvent and sample holder. The setup can be integrated within an MX beamline environment and exploits both absorption and phase contrast. The dose needed for the tomographic measurements could be low enough to allow the technique to be used for crystal integrity characterization and alignment. X-ray tomography has some major benefits compared with the optical-light-based crystal alignment protocols currently used.