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

Abstract

Native single-wavelength anomalous dispersion (SAD) is the most attractive de novo phasing method in macromolecular crystallography, as it directly utilizes intrinsic anomalous scattering from native crystals. However, the success of such an experiment depends on accurate measurements of the reflection intensities and therefore on careful data-collection protocols. Here, the low-dose, multiple-orientation data-collection protocol for native SAD phasing developed at beamline X06DA (PXIII) at the Swiss Light Source is reviewed, and its usage over the last four years on conventional crystals (>50 µm) is reported. Being experimentally very simple and fast, this method has gained popularity and has delivered 45 de novo structures to date (13 of which have been published). Native SAD is currently the primary choice for experimental phasing among X06DA users. The method can address challenging cases: here, native SAD phasing performed on a streptavidin–biotin crystal with P21 symmetry and a low Bijvoet ratio of 0.6% is highlighted. The use of intrinsic anomalous signals as sequence markers for model building and the assignment of ions is also briefly described.

Crystal structure of the leucine-rich repeat ectodomain of the plant immune receptor kinase SOBIR1

“…2a; see Section 2). The N-glycosylated protein was crystallized using the vapour-diffusion method and the structure was solved by MR-SAD on beamline X06DA at the Swiss Light Source (see Section 2 and Table 1; Basu et al, 2019). The solution in space group P6 5 comprises a dimer in the asymmetric unit, with the nine putative sulfur sites corresponding to a disulfide bridge in the N-terminal LRR capping domain, to a free cysteine and a methionine residue in the LRR core and to a free ion, which we interpreted as a chlorine anion originating from the crystallization buffer (Fig.…”

De novo crystal structure determination of double stranded RNA binding domain using only the sulfur anomalous diffraction in SAD phasing

“…Hence, as a compromise between higher anomalous signal at low energy and lower noise at high energy, sulfur-SAD phasing is usually carried out “off-edge” at shorter wavelengths (1.5–3 ​Å). Home-source with Cu (λ ​= ​1.54 ​Å) ( Sarma and Karplus, 2006 ) or Cr (λ ​= ​2.29 ​Å) radiations ( Watanabe, 2006 ) have been used to solve the structure of large and well-diffracting crystals, whereas synchrotron beamlines have been developed to optimize beam stability and minimize X-ray absorption effects for native SAD data collection in more difficult cases ( Doutch et al., 2012 ; Weinert et al., 2015 ; Wang et al., 2006 ; Basu et al., 2019a ; De Sanctis et al., 2016 ; Cianci et al., 2017 ). Moreover, the development of pixel array detectors that operate in single photon counting mode such as the Pilatus ( Broennimann et al., 2006 ) has been a breakthrough in macromolecular crystallography by enabling noise-free detection and novel data-acquisition modes.…”

“…Among the various synchrotron data-collection strategies that have been proposed to optimize native-SAD ( Olieric et al., 2016 ) - including sulfur-SAD - phasing, the use of a long-wavelength (2.7 ​Å) over 1.9 ​Å ( Cianci et al., 2016 ; Banerjee et al., 2016 ; Basu et al., 2019b ) was shown to be advantageous for 100 ​μm or smaller crystals ( Basu et al., 2019b ; Liebschner et al., 2016 ). Moreover, successful native SAD phasing was achieved in several difficult cases using low X-ray dose, high redundancy and multi-orientations data collection of multiple crystals ( Liu et al., 2012 , 2014 ; Assmann et al., 2020 ; Banerjee et al., 2016 ; Akey et al., 2014 ; El Omari et al., 2014 ; Klinke et al., 2015 ), or even a single crystal ( Weinert et al., 2015 ; Basu et al., 2019 , Basu et al., 2019 ), which obviously removes the problem of non-isomorphism between various crystals. The latest technical advances include solution-free mounting systems to minimize X-ray absorption at long-wavelengths ( Yu et al., 2020 ) and assembly of numerous partial single-crystal data sets from microcrystals with sizes less than 10 ​μm ( Cianci et al., 2019 ; Guo et al., 2019 ; Nass et al., 2020 ).…”

“…The latest technical advances include solution-free mounting systems to minimize X-ray absorption at long-wavelengths ( Yu et al., 2020 ) and assembly of numerous partial single-crystal data sets from microcrystals with sizes less than 10 ​μm ( Cianci et al., 2019 ; Guo et al., 2019 ; Nass et al., 2020 ). Despite these improvements, native SAD phasing is still not yet a routine method for successful structure solution ( Rose et al., 2015 ; Basu et al., 2019a ) because success is not only dependent on the content in anomalous scattering atoms but also on the crystal features, with a high diffracting power and high symmetry being advantageous.…”

“…In several cases, native SAD phasing was achieved using atoms other than S as scatterers, such as Ca, Cl, K, Na, Mg, which were constitutive of the protein or originated fortuitously from the crystallization solution ( Dauter et al., 1999 ; Weinert et al., 2015 ; Olieric et al., 2016 ; Basu et al., 2019a , 2019b ; Mueller-Dieckmann et al., 2007 ). Because the anomalous scattering factors f’’ of the atoms that are heavier than S (Ca, Cl, K) are higher than that of sulfur in the 1–2 ​Å wavelength range used at synchrotrons (see Fig.…”