However, both MR and SAD phasing can fail for unavoidable reasons, and phasing by multiple isomorphous replacement (MIR Green et al., 1954 ▸ Blow & Crick, 1959 ▸), multiple-wavelength anomalous dispersion (MAD Hendrickson, 2014 ▸ Hendrickson & Teeter, 1981 ▸) or multiple isomorphous replacement with anomalous scattering (MIRAS Vonrhein et al., 2007 ▸ Rossmann, 1961 ▸) are alternatives to MR and SAD that should be included in broader phasing strategies. Most of the structures deposited in the PDB are currently phased by one or the other of these two methods (Burley et al., 2019 ▸). The focus of our developments has been phasing by molecular replacement (MR Huber, 1965 ▸ Read, 2001 ▸) and single-wavelength anomalous dispersion (SAD Hendrickson & Teeter, 1981 ▸ Pannu & Read, 2004 ▸) because these methods are similar in having the relative ease of requiring only a single (merged) data set, because they are both amenable to rigorous likelihood treatments and because single-wavelength data collection can require a lower total radiation dose than multiple-wavelength methods.
Our Phaser crystallographic software for phasing macromolecular crystal structures based on maximum likelihood and multivariate statistics (Bricogne, 1992 ▸, 1997 ▸ Read, 2001 ▸) has been an asset to the crystallographic community, having solved tens of thousands of macromolecular crystal structures in the Protein Data Bank (PDB Burley et al., 2019 ▸). The description of the phasing strategy with directed acyclic graphs is a generalization that extends beyond the functionality of Phasertng, as it can incorporate results from bioinformatics and other crystallographic tools, and will facilitate multifaceted search strategies, dynamic ranking of alternative search pathways and the exploitation of machine learning to further improve phasing strategies. As a first application of phasertng, its advantages are demonstrated in the context of phasertng.xtricorder, a tool to analyse and triage merged data in preparation for molecular replacement or experimental phasing. In Phasertng, the codebase of Phaser has been rebuilt, with an emphasis on modularity, on scripting, on speed and on continuing algorithm development. To this end, the development of new software, Phasertng, which uses directed acyclic graphs natively for input/output, has been initiated. The crystallographic software supporting the graph data structure must be strictly modular so that nodes in the graph are efficiently generated by the encapsulated functionality.
Describing phasing in terms of a directed acyclic graph allows graph-management software to track and manage the path to structure solution. Accelerating this move has been improvements in phasing methods, which are now able to extract phase information from the placement of very small fragments of structure, from weak experimental phasing signal or from combinations of molecular replacement and experimental phasing information.
Crystallographic phasing strategies increasingly require the exploration and ranking of many hypotheses about the number, types and positions of atoms, molecules and/or molecular fragments in the unit cell, each with only a small chance of being correct.