The gateway to X-ray crystallography is the growth of macromolecular crystals. An attempt is made here to acquaint investigators with the principles and practice of protein crystallization, some recent innovations, and to touch upon some of the remaining problems.

Approaches to automated and robot-assisted harvesting of protein crystals are critically reviewed. While no true turn-key solutions for automation of protein crystal harvesting are currently available, systems incorporating advanced robotics and micro-electromechanical systems represent exciting developments with the potential to revolutionize the way in which protein crystals are harvested.

The properties of the mesoscopic protein-rich clusters, within which protein crystal nuclei form, are reviewed. The cluster populations occupy from 10⁻⁷ to 10⁻³ of the solution volume; the cluster radii are of order 100 nm; the clusters exist owing to the formation of transient oligomers that, for some proteins, may be due to conformational flexibility.

New imaging techniques, particularly AFM, permitted the elucidation of the mechanisms for protein and virus crystal growth. They have also allowed direct visualization of crystal defect structure and the consequences of impurity incorporation.

This article highlights some of the ground-based studies emanating from NASA's Microgravity Protein Crystal Growth (PCG) program, and includes a more detailed discussion of the history and the progress made in one of the NASA-funded PCG investigations involving the use of measured second virial coefficients (B values) as a diagnostic indicator of solution conditions conducive to protein crystallization.

Automation is the response to overcoming the crystallization bottleneck in biological crystallography. This review provides a summary of the current methods and technologies applied in automated platforms for the setup of initial and follow-up crystallization experiments.

The rich history of crystallization and how that history influences current practices is described. The tremendous impact of crystallization screens on the field is discussed.

As technology advances, the crystal volume that can be used to collect useful X-ray diffraction data decreases. The technologies available to detect and study growing crystals beyond the optical resolution limit and methods to successfully place the crystal into the X-ray beam are discussed.

Microseed matrix screening is increasingly successful in the optimization of protein crystallization leads.

The PDB is looked at to find data to answer the question `How well do our current screens cover crystallization space?' and to try to find answers to the more general question about the most efficient strategy to employ in a crystallization campaign.

Strategies and procedures for the optimization of macromolecular crystal-growth conditions are surveyed. The objective of optimization is to grow crystals with the greatest degree of perfection and that yield the most accurate and precise X-ray diffraction data.

In the last three decades, membrane-protein crystallization has changed from overwhelmingly difficult to nearly routine. This review offers a snapshot of the current state of the art, focusing particularly on the role of detergents in this process.

A comprehensive and up-to-date review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes is reported. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described.

Fluorescence, whether intrinsic or by using trace fluorescent labeling, can be a powerful aid in macromolecule crystallization. Its use in screening for crystals is discussed here.

While crystallization phase diagrams are excellent tools to conceptualize phase relations in protein solutions, they need to be used appropriately and to be compatible with their physicochemical meaning.

Growing large-volume crystals are necessary and important for neutron macromolecular crystallography. Experimental strategies, techniques and theoretical considerations for protein crystal growth are reviewed.

Current methods, reagents and experimental hardware for successfully and reproducibly flash-cooling macromolecular crystals to cryogenic temperatures for X-ray diffraction data collection are reviewed.

In vivo formation of protein crystals and their isolation and structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources is described.

An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer.

This article reviews the role of mass transport in protein-crystallization experiments.