The elucidation of protein structures by X-ray crystallography remains the most effectual method to provide accurate structural details at atomic resolution for rational drug design and other biotechnological research studies. Also, emerging applications of protein crystals as ordered nanostructure scaffolds for catalysis, imaging, and drug delivery are attracting much attention. However, the first step of these applications is obtaining high-quality crystals, which is still an obstacle. Successful crystallization requires two steps: nucleation and crystal growth, while the nucleation is a precondition for harvesting the crystal of interest. So controlling protein nucleation may be an alternative breakthrough for this bottleneck. It is well known that nucleants can induce protein crystallization and improve crystal quality, so investigation on the nucleants that can be universally used for any protein crystallization is ongoing. This manuscript reviews the advances that have been achieved using nucleants in protein crystallization and it is a suitable reference for practical crystallization.

Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.

A magnetic field has been proved useful in protein crystallization in that it can help to improve the crystal quality, which is essential for high-resolution diffraction using crystallography. However, it is not widely utilized as a high-field magnet usually provides limited space which is usually too small for commercially available crystallization plates, so that the crystallization screening and optimization in such a magnet become a challenging task. Here we show that a specially designed flexible capillary array containing 96 conditions can be easily crimped into a small curl so that it can be accommodated into the bore of a large gradient superconducting magnet. Using the capillary array, we conducted a systematic investigation on the crystallization screening and crystal quality of lysozyme under different simulated gravities (μg, 1g, and 2g) in a large-gradient magnetic field and normal gravity outside the magnet. This is the first statistical study of the crystallization screening of protein in such a high-field magnet. The results showed that both the simulated gravity and the magnetic field affect the crystallization of lysozyme. The simulated microgravity can significantly improve the crystal quality, and the simulated hypergravity is helpful to increase the chance for obtaining crystallization conditions. Moreover, the method exhibits more potential features, such as the capability of in situ diffraction and the applicability for space experiments.

Application of protein crystallization methodologies to screen optimal solution formulations for proteins prone to aggregation.