Providing fundamental information on intra/intermolecular interactions and physicochemical properties, the three-dimensional structural characterization of biological macromolecules is of extreme importance towards understanding their mechanism of action. Among other methods, X-ray powder diffraction (XRPD) has proved its applicability and efficiency in numerous studies of different materials. Owing to recent methodological advances, this method is now considered a respectable tool for identifying macromolecular phase transitions, quantitative analysis, and determining structural modifications of samples ranging from small organics to full-length proteins. An overview of the XRPD applications and recent improvements related to the study of challenging macromolecules and peptides toward structure-based drug design is discussed. This review congregates recent studies in the field of drug formulation and delivery processes, as well as in polymorph identification and the effect of ligands and environmental conditions upon crystal characteristics. These studies further manifest the efficiency of protein XRPD for quick and accurate preliminary structural characterization.

Crystallization of recombinant proteins in living cells is an exciting new approach for structural biology that provides an alternative to the time-consuming optimization of protein purification and extensive crystal screening steps. Exploiting the potential of this approach requires a more detailed understanding of the cellular processes involved and versatile screening strategies for crystals in a cell culture. Particularly if the target protein forms crystalline structures of unknown morphology only in a small fraction of cells, their detection by applying standard visualization techniques can be time consuming and difficult owing to the environmental challenges imposed by the living cells. In this study, a high-brilliance and low-background bioSAXS beamline is employed for rapid and sensitive detection of protein microcrystals grown within insect cells. On the basis of the presence of Bragg peaks in the recorded small-angle X-ray scattering profiles, it is possible to assess within seconds whether a cell culture contains microcrystals, even in a small percentage of cells. Since such information cannot be obtained by other established detection methods in this time frame, this screening approach has the potential to overcome one of the bottlenecks of intracellular crystal detection. Moreover, the association of the Bragg peak positions in the scattering curves with the unit-cell composition of the protein crystals raises the possibility of investigating the impact of environmental conditions on the crystal structure of the intracellular protein crystals. This information provides valuable insights helping to further understand the in cellulo crystallization process.

The polymorphism of human insulin upon pH variation was characterized via X-ray powder diffraction, employing a crystallization protocol previously established for co-crystallization with phenolic derivatives. Two distinct rhombohedral (R3) polymorphs and one cubic (I2₁3) polymorph were identified with increasing pH, corresponding to the T₆, T₃R₃ ^f and T₂ conformations of insulin, respectively. The structure of the cubic T₂ polymorph was determined via multi-profile stereochemically restrained Rietveld refinement at 2.7 Å resolution. This constitutes the first cubic insulin structure to be determined from crystals grown in the presence of zinc ions, although no zinc binding was observed. The differences of the polycrystalline variant from other cubic insulin structures, as well as the nature of the pH-driven phase transitions, are discussed in detail.