Real-time monitoring of large-scale crystal growth using batch crystallization for serial crystallography

Serial crystallography (SX) enable to visualize the molecular dynamics of macromolecule at room temperature with minimal radiation damage. Large-scale batch crystallization method is widely applied for the SX sample preparation, but the simultaneous growth of multiple crystals in the batch is not well-studied. In this study, we report the real-time monitoring of large-scale crystal growth during batch crystallization using the protein lysozyme. Highly concentrated lysozyme immediately formed random-sized aggregates upon mixing with the crystallization solution. The nucleation and crystal growth started from the aggregation area. In regions with high aggregation density, the size of initially grown crystals increased rather than further nucleation and crystal formation. The qualities of the crystals obtained from the large-scale batch crystallization method were verified by serial synchrotron crystallography, which determined the room-temperature structure of lysozyme at 1.6 angstrom. Based on the activation energy and nucleation barrier, our results indicate that a high concentration of lysozyme initially forms non-specific aggregates, a process that involves nucleation from non-specific aggregates. Then, the nonspecifically aggregated proteins nucleate to form crystals that grow into larger crystals. The findings of this study provide a better understanding of the phenomenon of a large number of crystal growths in large-scale batch crystallization.

Automated summary

Serial crystallography (SX) has been used to visualize the molecular dynamics of macromolecules at room temperature with minimal radiation damage. However, the simultaneous growth of multiple crystals in large-scale batch crystallization has not been well-studied. In this study, the real-time monitoring of large-scale crystal growth during batch crystallization was reported using lysozyme as a model protein. The findings contribute to a better understanding of the phenomenon of multiple crystal growths in large-scale batch crystallization.