Structure solution methods address the phase problem in X-ray crystallography — determining the phases of diffraction reflections that are lost during measurement. Three major approaches exist: molecular replacement (MR) uses a known homologous structure as a search model, rotating and translating it to find the correct orientation and position in the new crystal's unit cell; isomorphous replacement (MIR/SIR) introduces heavy atoms into the crystal and uses the intensity differences between native and derivative data sets to calculate phases; and anomalous dispersion (SAD/MAD) exploits the wavelength-dependent scattering of specific atoms (selenium, introduced via selenomethionine labeling) to extract phase information from a single crystal form. Molecular replacement dominates current practice because of the large number of known structures available as search models.
The phase problem is the central obstacle in X-ray crystallography: you measure intensities but need phases to calculate the electron density map. Three strategies have been developed to overcome it, each exploiting a different physical or biological principle.
Molecular replacement (MR) is the most widely used method. If the structure of a homologous protein is known, it can serve as an initial estimate of the target's structure. The search model is systematically rotated (rotation function) and translated (translation function) to find the orientation and position within the target crystal's unit cell that best explains the observed diffraction data. Once correctly placed, the model provides approximate phases. These approximate phases, combined with the experimental amplitudes, generate an electron density map that shows features of the target not present in the search model. Iterative cycles of model building and refinement improve the phases until the structure converges. MR is fast and requires no special crystal preparation, but it fails when no suitable homolog exists.
Isomorphous replacement was the method used to solve the first protein crystal structures (myoglobin, hemoglobin). Heavy atoms (mercury, platinum, gold) are soaked into the crystal, binding at specific sites without disrupting the crystal lattice (isomorphous = same crystal form). The intensity differences between native and heavy-atom-derivative diffraction patterns are attributable to the heavy atoms, whose positions can be determined. These positions provide the initial phase estimates. Multiple isomorphous replacement (MIR) uses two or more derivatives to resolve the phase ambiguity. The method is elegant in principle but laborious in practice — finding derivatives that bind without disrupting the crystal, collecting multiple complete data sets — which is why it has been largely superseded by anomalous methods.
Anomalous dispersion exploits the fact that atoms scatter X-rays differently near their absorption edges. Selenium (incorporated as selenomethionine), sulfur (in native cysteine/methionine), or metal ions absorb X-rays at specific wavelengths, producing a small anomalous scattering signal that breaks the symmetry of the diffraction pattern. In SAD (single-wavelength anomalous dispersion), data is collected at one wavelength near the absorption edge, and the anomalous differences reveal the positions of the anomalous scatterers (selenium atoms). These positions provide phase estimates, which are improved by density modification (solvent flattening, histogram matching). In MAD (multi-wavelength anomalous dispersion), data at multiple wavelengths provides additional constraints for more accurate phasing. Selenomethionine SAD has become the default experimental phasing method because it combines straightforward sample preparation (biosynthetic incorporation), strong anomalous signal, and robust computational methods — making it practical for even moderately diffracting crystals.