Date Published: March 01, 2019
Publisher: International Union of Crystallography
Author(s): Shibom Basu, Aaron Finke, Laura Vera, Meitian Wang, Vincent Olieric.
A broadly applicable, simple and fast data-collection strategy for native SAD is described, which has become the primary choice for experimental phasing among users of the macromolecular crystallography beamline X06DA (PXIII) at the Swiss Light Source. Its usage over the last four years is reviewed.
Single-wavelength anomalous dispersion (SAD) is the most popular experimental phasing technique for de novo structure determination by macromolecular crystallography (MX). Nearly 50% of the novel structures deposited in the Protein Data Bank (PDB; Berman et al., 2000 ▸) were determined using this method. The vast majority of these SAD experiments exploited the strong anomalous signal of heavy atoms, delivered either by soaking of heavy elements or by the expression of selenomethionyl protein (Hendrickson, 2014 ▸). However, these derivatizations can be problematic owing to loss of both isomorphism and diffraction. Thus, a SAD experiment using only native crystals is very appealing. This method, called native SAD, which exploits the weak anomalous signals from light elements (Z < 20, i.e. mostly S, P, Cl−, K+ and Ca2+) that are natively present in biomolecules, has certain challenges that have, until recently, kept it from being a general method. The anomalous signal of these light scatterers is higher at low energies (<6 keV), but X-ray absorption from the sample, including the crystal, the surrounding solvent and the mounting medium, and especially the absorption of X-rays by air lead to a significant attenuation of the diffracted signals. Other difficulties in low-energy crystallography are on the detector side, with both inaccurate calibration and a loss of high-angle diffraction with a typical flat configuration, owing to the increasing Bragg angles of reflection. To overcome these limitations and push the limit of native SAD phasing (Bent et al., 2016 ▸), special setups designed for native SAD data collection at energies below 5 keV have been developed at beamlines I23 at Diamond Light Source in the UK (Wagner et al., 2016 ▸) and BL-1A at the KEK Photon Factory in Japan (Liebschner et al., 2016 ▸). Provided that diffraction extends to ∼2.8 Å resolution, high-quality data for native SAD phasing can also be obtained on conventional macromolecular crystallography beamlines using an energy of 6 keV (Mueller-Dieckmann et al., 2007 ▸; Liu et al., 2012 ▸; Weinert et al., 2015 ▸). We and our users have routinely been using our native SAD strategy for the last four years, and we have already reported some of our major successes (Weinert et al., 2015 ▸; Olieric et al., 2016 ▸). Based on our archive (November 2014 to January 2019), which reflects only the projects that we followed with users, we were able to determine 75 native SAD structures out of a total of 97 attempted cases. This includes the successful determination of 45 de novo structures (Fig. 2 ▸), of which 13 have already been published (Campagne et al., 2014 ▸; Goncharenko et al., 2015 ▸; Niesser et al., 2016 ▸; Brandmann & Jinek, 2015 ▸; Jiang et al., 2017 ▸; Ou et al., 2017 ▸; Leonaitė et al., 2017 ▸; Śledź & Jinek, 2016 ▸; Hermanns et al., 2018 ▸; Scietti et al., 2018 ▸; Hohmann et al., 2018 ▸; Liu et al., 2019 ▸; Wang et al., 2019 ▸). Of those 97 cases, 27 structures (shown as ‘method development’ in Fig. 2 ▸) were solved to demonstrate the potential of our native SAD strategy. The majority of the 75 solved structures, provided by various users, are of high symmetry (orthorhombic or higher), are well diffracting (< 2.5 Å resolution) and are relatively small (<100 kDa). For such cases, a routine solution required an average of 4.5 × 360° ω scans at varying orientations and was completed in 15 min or less. In the past four years, we have developed and built up a low-dose, multiple-orientation data-collection strategy for native SAD from a niche technique into a standard one that any user can easily accomplish in under 20 min. Being fast and experimentally very simple, X06DA users routinely perform experiments at 6 keV during their assigned beam time. Indeed, neither changes in the experimental setup nor prior knowledge of the crystal system are required. Advances in beamline instrumentation, in particular a highly precise multi-axis goniometer and fast photon-counting detectors, enabled these native SAD experiments. While our native SAD data-collection strategy is readily available at beamlines equipped with multi-axis goniometers, such as the popular mini-kappa (Brockhauser et al., 2011 ▸), new multi-axis goniometers such as SmarGon (the multi-axis goniometer from SmarAct GmbH) are being developed and will allow full automation in multi-orientation data collection. The broad applicability, simplicity and rapidity of our strategy should encourage the structural biology community to attempt native SAD on their own as a first choice for experimental phasing: the chance of success is high, here of the order of 75–80%. Source: http://doi.org/10.1107/S2059798319003103