High-throughput X-ray crystallography

Abstract

Structural-genomics projects have contributed to major developments in high-throughput (HT) X-ray crystallography. Various steps involved in determining an X-ray crystal structure can be automated and miniaturized, including cloning, protein expression and purification, crystallization, and X-ray data collection. Many of the protocols developed in HT structural genomics for the generation of multiple protein targets can also be adapted for crystallization of challenging proteins. In particular, multiple constructs of a single protein target can be produced in parallel to find a construct that is amenable to crystallization. This chapter summarizes recent developments in HT X-ray crystallography, and describes how these approaches can be adapted in individual research laboratories to obtain suitable protein constructs for subsequent X-ray structure determination. Keywords: high-throughput crystallography; automation; protein engineering; fusion proteins; promotion of crystallization; ligation-independent cloning; recombination cloning; autoinduction; high-throughput cloning; high-throughput protein expression; high-throughput crystallization; crystallization robots; automated crystal mounting