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

Maxim Polikarpov,Anatoly Snigirev,Irina Snigireva,Gleb Bourenkov,Krisztian Csanko,Sophie Zimmermann,Sandor Brockhauser,Thomas R Schneider

doi:10.1107/s2059798319011379

Maxim Polikarpov, Anatoly Snigirev + Show 6 more

Open Access

https://doi.org/10.1107/s2059798319011379

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Abstract

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.

Highlights

A crucial step in setting up a successful X-ray diffraction experiment is the accurate centering of the crystal of interest with respect to the X-ray beam
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 phasecontrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix
We demonstrate that the X-ray beam available on the macromolecular crystallography beamline P14 at PETRA III at DESY, Hamburg, Germany is of sufficient quality for imaging protein crystals at micrometre resolution with requirements in dose and wall-clock time that are compatible with crystallographic data collection from the same sample

Summary

Introduction

A crucial step in setting up a successful X-ray diffraction experiment is the accurate centering of the crystal of interest with respect to the X-ray beam. These procedures have proven to be useful for diffraction data collection from crystals grown and mounted in lipidic cubic phase (LCP; Landau & Rosenbusch, 1996; Caffrey, 2000) or in meso phase (Caffrey, 2003) and subsequently cryocooled. The entire tolerable dose may be needed to acquire a single interpretable diffraction pattern, implying the use of serial crystallography approaches (Gati et al, 2014)

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