Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

Abstract

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicator R(anom)/R(p.i.m.), an inconsistency in the documentation of the anomalous R factor is noted and reported.

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

“…Higher I/ usually relates well to better merging factors, which may explain this trend. Additionally, R meas may increase at 2.7 Å wavelength because of systematic errors owing to absorption, as was observed by Weiss and coworkers for data collected at 2.583 Å wavelength (Weiss et al, 2001) and 2.65 Å wavelength (Gorgel et al, 2015). Lastly, R meas is calculated with merged I + /I À .…”

Over-Expression, Secondary Structure Characterization, and Preliminary X-ray Crystallographic Analysis of Xenopus tropicalis Ependymin

“…In the future, we plan to improve the current 2.7 Å resolution crystal for better diffraction, and determine the high resolution structure of frog EPN by direct phasing. During the process, we plan to exploit conventional metal soaking [22] and other well-known techniques to work such as the use of (Ta 6 Br 12 ) 2+ [23] or 5-amino-2,4,6-triiodoisophthalic acid [24], and even sulfur-SAD [25,26] or ultraviolet radiation damage-induced phasing (UV-RIP) [27] methods. Since no structural information on any EPN exists in databases, the structure would give us insight into understanding its function.…”

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

“…Here, we review the recent cases, described in the literature, where native SAD phasing was used successfully using no atoms heavier than sulfur as anomalous scatterers ( Table 1 ). Difficult cases include crystals belonging to low symmetry space groups ( Basu et al., 2019a ; Banerjee et al., 2016 ; Klinke et al., 2015 ), diffracting at poor resolution ( Liu et al., 2014 ; Cianci et al., 2016 ; Basu et al., 2019b ; Akey et al., 2014 ; El Omari et al., 2014 ; Klinke et al., 2015 ) with an extremely low sulfur content ( Basu et al., 2019a ; Gorgel et al., 2015 ) or large substructure ( Basu et al., 2019b ; Akey et al., 2014 ; Klinke et al., 2015 ). We also report our experience, where several data sets collected in various goniometer orientations on a single well-diffracting crystal of the double stranded RNA binding domain (dsRBD) of human dihydrouridine synthase hdus2 were used for successful sulfur-SAD phasing.…”

“…Examples of other structures solved by native SAD using sulfur and another scattering atom can be found in Table 1 of Olczak and Cianci, 2018 and Supplementary Tables 1 of Weinert et al. (2015) and Gorgel et al. (2015) .…”