Solid/liquid interface induced protein crystallization

X-ray crystallography remains the gold standard for resolving high-resolution atomic structures of biomolecules. Its unparalleled precision continues to provide critical structural insights that drive advances in drug discovery, enzyme mechanism elucidation, and molecular engineering across biotechnology, materials science, and nanomedicine. Despite its strengths, its success is fundamentally limited by the requirement for high-quality, well-ordered crystals, a persistent bottleneck in structural biology. Crystallization begins with nucleation, the critical step where solute molecules organize into a stable nucleus capable of initiating crystal growth. Controlling nucleation is essential for improving crystal reproducibility, size, and diffraction quality. To overcome this challenge, various interfaces, including liquid/liquid, air/water, and solid/liquid, have been explored, with the solid/liquid interface gaining increasing attention due to its ability to promote and modulate nucleation events. This review systematically discusses strategies utilizing solid/liquid interfaces to enhance protein crystallization efficiency and quality. It emphasizes the roles of diverse surfaces, including porous, hydrophobic, charged, rough, and functionalized substrates, and additive-assisted nucleation using micro-/macroparticles, nanoparticles, and DNA. Both electrostatic and non-electrostatic surface-induced mechanisms are critically analysed, with mechanistic insights into how these surfaces influence nucleation kinetics and crystal growth mechanisms. Comparative evaluations of different surface and additive systems are presented to identify effective nucleation enhancers and promote rational crystallization design. By deepening our understanding of interface-mediated nucleation and growth, this review provides a comprehensive knowledge base to support the rational development of reproducible, high-throughput crystallization strategies and outlines future directions for innovation in structural biology and crystallization science.