On the influence of crystal size and wavelength on native SAD phasing

Abstract

Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g. air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.

Crystallographic analysis of murine p24γ2 Golgi dynamics domain

“…We aimed to determine the crystal structure of mouse p24γ2 GOLD domain by the single anomalous diffraction method using intrinsic sulfur atoms . Although the resolution of the dataset is moderate, we could detect the subtle anomalous signals originating from the sulfur atoms using an efficient merging technique.…”

“…X‐ray diffraction datasets for phase determination were collected at the beamline BL‐1A in the Photon Factory (Tsukuba, Japan) . Phase determination was carried out by the single anomalous dispersion (SAD) method using intrinsic sulfur atoms.…”

Development of low-energy X-ray detectors using LGAD sensors

“…However, many synchrotron experiments are performed in the soft X-ray energy range (Hitchcock, 2015) due to the higher cross section for thin or low-interacting samples. Furthermore, at low energies, the K-edges of many light elements, which are useful for example in macromolecular crystallography (Liebschner et al, 2016), are present, as well as the L-edges of 3D transition metals which are relevant for studying copperbased superconductors or magnetic structures by means of ISSN 1600-5775 # 2019 International Union of Crystallography scanning X-ray transmission microscopy (STXM), ptychography or resonant diffraction (Fink et al, 2013). Hybrid detectors have not yet been applied to soft-energy X-rays due to their noise.…”

Crystal structure of the nitrosuccinate lyase CreD in complex with fumarate provides insights into the catalytic mechanism for nitrous acid elimination

“…Data collection with an X-ray of wavelength 1.90 A enabled us to use anomalous effects of sulfur atoms for phasing [37]. Data collection with an X-ray of wavelength 1.90 A enabled us to use anomalous effects of sulfur atoms for phasing [37].…”