Long-wavelength native-SAD phasing: opportunities and challenges

Abstract

Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z < 20). The anomalous signal of sulfur in particular, is enhanced at long wavelengths, however the absorption of diffracted X-rays owing to the crystal, the sample support and air affects the recorded intensities. Thereby, the optimal measurable anomalous signals primarily depend on the counterplay of the absorption and the anomalous scattering factor at a given X-ray wavelength. Here, the benefit of using a wavelength of 2.7 over 1.9 Å is demonstrated for native-SAD phasing on a 266 kDa multiprotein-ligand tubulin complex (T2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.

Making routine native SAD a reality: lessons from beamline X06DA at the Swiss Light Source

“…Upon considering lower X-ray energies for native SAD, X-ray absorption owing to crystal thickness is a major issue (Arndt, 1984;Wagner et al, 2016;Liebschner et al, 2016). This can be overcome with spherical laser-shaped crystals (Kitano et al, 2004;Watanabe, 2006;Basu, Olieric et al, 2019) or with micrometre-sized crystals, which are now routinely used in the field of structural biology. High-multiplicity data are challenging to obtain from micrometre-sized (<30 mm) single crystals, but it has been shown that accurate native SAD data can be gleaned from such crystals at the synchrotron using serial crystallography approaches (Huang et al, 2015(Huang et al, , 2016(Huang et al, , 2018Diederichs & Wang, 2017;Weinert et al, 2017).…”

A shared vision for macromolecular crystallography over the next five years

“…While beamline I23 at DLS serves as a prototype, simplifications to its design are possible. Beamline 1A at Photon Factory operates in a helium atmosphere and sports two stock HPC detectors that can be arranged at a 25° angle with respect to each other (Basu et al , 2019). No matter the beamline specifics, low-energy MX is a decidedly low-throughput application where difficult problems with high impacts will be solved.…”

PyMDA: microcrystal data assembly using Python

“…Compared with XFELs which produce only one diffraction pattern for every microcrystal, synchrotron microdiffraction beamlines are optimized for collection of a small wedge of rotation data from each microcrystal, thus greatly improving data quality from microcrystals (Smith et al, 2012;Fuchs et al, 2016;Yamamoto et al, 2017). With the implementation of new data collection methods, microcrystal data acquisition at synchrotrons is now routine and maturing (Gati et al, 2014;Coquelle et al, 2015;Zander et al, 2015;Diederichs & Wang, 2017;Owen et al, 2017;Sanishvili & Fischetti, 2017;Gao et al, 2018;Huang et al, 2018;Basu et al, 2019;Cianci et al, 2019;Dauter, 2019;Guo et al, 2019).…”

“…In addition, microcrystals are affected by environmental changes and their unit cells may have large variations. To rationally treat unit-cell variations, radiation damage and incomplete data in microcrystals, we and others have developed data assembly workflows (Guo et al, 2018(Guo et al, , 2019Yamashita et al, 2018;Basu et al, 2019;Cianci et al, 2019). With our workflow, we were able to assemble anomalous diffraction data from about 1200 native microcrystals and used the data for a successful single-wavelength anomalous diffraction analysis (Guo et al, 2019).…”