Use of massively multiple merged data for low-resolution S-SAD phasing and refinement of flavivirus NS1

Abstract

An emergent challenge in macromolecular crystallography is the identification of the substructure from native anomalous scatterers in crystals that diffract to low to moderate resolution. Increasing the multiplicity of data sets has been shown to make previously intractable phasing problems solvable and to increase the useful resolution in model refinement. For the West Nile virus nonstructural protein 1 (NS1), a protein of novel fold, the utility of exceptionally high multiplicity data is demonstrated both in solving the crystal structure from the anomalous scattering of the native S atoms and in extending the useful limits of resolution during refinement. A high-multiplicity data set from 18 crystals had sufficient anomalous signal to identify sulfur sites using data to 5.2 Å resolution. Phases calculated to 4.5 Å resolution and extended to 3.0 Å resolution were of sufficient quality for automated building of three-quarters of the final structure. Crystallographic refinement to 2.9 Å resolution proceeded smoothly, justifying the increase in resolution that was made possible by combining multiple data sets. The identification and exclusion of data from outlier crystals is shown to result in more robust substructure determination.

Assessing and maximizing data quality in macromolecular crystallography

“…The calculations have been built into the PHENIX system [9], and already been used in some reports (e.g. [10•, 11••]). While how to best use this information in guiding and validating refinements is not yet clear, it provides a welcome replacement for the practice of comparing refinement R-factors with data reduction R-factors (e.g.…”

“…Selecting a more generous high resolution cutoff (e.g. [11••, 16•]) and/or increasing the multiplicity of the high resolution data (e.g. [14]), makes the ‘overall’ statistics become worse even though the resulting data are better.…”

Facilitating best practices in collecting anomalous scattering data forde novostructure solution at the ESRF Structural Biology Beamlines

“…3b) and, if necessary, modify the parameters of the proposed strategy. For experiments aimed at de novo structure solution, the collection of highly redundant data is often advisable, and this becomes even more relevant when weak anomalous signals are to be exploited (Dauter & Adamiak, 2001;Cianci et al, 2008;Akey et al, 2014;Weinert et al, 2015;Liu & Hendrickson, 2015).…”

“…The two data sets sets are processed separately and then scaled together to provide the final data set. Although, to our knowledge a systematic study on the beneficial use of inverse-beam geometry is not available, many examples of successful phasing experiments using this technique have been reported in the literature (Liu et al, 2012(Liu et al, , 2013Akey et al, 2014).…”

Progress in protein crystallography

“…Indeed, currently most of the novel structures are solved by SAD. Moreover, the small anomalous signal of the intrinsically present atoms of sulfur or phosphorus in native macromolecules is also gaining popularity [61], even with data measured and merged from several crystals [62, 63]. …”