Visualization of protein crystals by high-energy phase-contrast X-ray imaging.

For the extraction of the best possible X-ray diffraction data from macromolecular crystals, accurate positioning of the crystals with respect to the X-ray beam is crucial. In addition, information about the shape and internal defects of crystals allows the optimization of data-collection strategies. Here, it is demonstrated that the X-ray beam available on the macromolecular crystallography beamline P14 at the high-brilliance synchrotron-radiation source PETRA III at DESY, Hamburg, Germany can be used for high-energy phase-contrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix. Three-dimensional tomograms have been obtained at X-ray doses that are substantially smaller and on time scales that are substantially shorter than those used for diffraction-scanning approaches that display protein crystals at micrometre resolution. Adding a compound refractive lens as an objective to the imaging setup, two-dimensional imaging at sub-micrometre resolution has been achieved. All experiments were performed on a standard macromolecular crystallography beamline and are compatible with standard diffraction data-collection workflows and apparatus. Phase-contrast X-ray imaging of macromolecular crystals could find wide application at existing and upcoming low-emittance synchrotron-radiation sources.

Calibration of rotation axes for multi-axis goniometers in macromolecular crystallography.

The installation of multi-axis goniometers such as the ESRF/EMBL miniKappa goniometer system has allowed the increased use of sample reorientation in macromolecular crystallography. Old and newly appearing data collection methods require precision and accuracy in crystal reorientation. The proper use of such multi-axis systems has necessitated the development of rapid and easy to perform methods for establishing and evaluating device calibration. A new diffraction-based method meeting these criteria has been developed for the calibration of the motors responsible for rotational motion. This method takes advantage of crystal symmetry by comparing the orientations of a sample rotated about a given axis and checking that the magnitude of the real rotation fits the calculated angle between these two orientations. Hence, the accuracy and precision of rotational motion can be assessed. This rotation calibration procedure has been performed on several beamlines at the ESRF and other synchrotrons. Some resulting data are presented here for reference.