Abstract
During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.
Highlights
Serial femtosecond crystallography (SFX) at X-ray beam at a free-electron laser (XFEL) radiation sources and serial crystallography at synchrotron-radiation sources are relatively new approaches for the collection of diffraction data for the structure determination of biological macromolecules
Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electrondensity maps that were sufficient for building a complete structural model of proteinase K at 1.9 Aresolution using automatic model-building tools
We report here the first time that SIRAS has successfully been applied to serially collected data at a synchrotron-radiation source, which could be directly compared with the application of SIRAS phasing to SFX data
Summary
Serial femtosecond crystallography (SFX) at XFEL radiation sources and serial crystallography at synchrotron-radiation sources (serial millisecond crystallography; SMX) are relatively new approaches for the collection of diffraction data for the structure determination of biological macromolecules. Systematic inaccuracies and variances in the data resulting from experimental factors, such as the wide spectral distribution of the XFEL as well as changes in the sample-to-detector distance when exchanging the sample-delivery nozzle and variations in the size of the microcrystal distribution or liquid-jet width, have been shown to severely hamper phasing attempts using heavy-atom data sets (Nass et al, 2016) This has so far been overcome by using a large number of diffraction patterns, and Yamashita et al (2015) demonstrated that single-wavelength anomalous diffraction (SAD) phasing with approximately 80 000 patterns as well as single isomorphous replacement with anomalous scattering (SIRAS) phasing with approximately 20 000 patterns were feasible. They showed that 12 000 patterns were sufficient for successful SIRAS phasing, and a further reduction in the number of images required was achieved by a combination of single isomorphous replacement (SIR) and SIRAS (Nakane et al, 2016)
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