Facing the phase problem.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we - myself, my laboratory and the diffraction community - have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.