Calculating an electron density map, into which the structural model will be built, requires phase information. X-ray detectors register only the intensities of the diffracted waves; the phase information is lost during this process and must be retrieved using other methods. Currently, heavy-atom replacement is rarely used. Instead, molecular replacement is the most popular method. Molecular replacement requires an experimental 3D structure of the protein or a close homolog (typically with sequence identity above 40-50%) to calculate initial phases and an electron density map. At times, computer-modeled structures (e.g., from AlphaFold) may also be used to solve the phase problem. However, this requires that the conformation of the computed structure (especially for multidomain proteins) match that of the crystal structure being studied. If molecular replacement fails, we must use de novo structure determination. For this, typically, a selenomethionine (SeMet)-labeled protein sample, which we can produce upon the client's request, enables structure determination using synchrotron radiation and anomalous X-ray scattering. Crystallographic software packages Phaser and Molrep are typically used to phase macromolecular crystal structures using molecular replacement and single-wavelength anomalous dispersion.
