Production and Crystallization of Bacterial Type V Secretion Proteins

X-ray crystallography has become the most powerful approach to determine the three dimensional structures of proteins. The major bottleneck issues in protein crystallography are the availability of high-quality protein samples and the production of diffracting crystals. Since the type V secretion pathway involves unusually large substrate proteins (passenger domains or TpsA) and membrane proteins (β-barrel domains or TpsB), crystallography of type V secretion proteins deals with additional challenges in protein production and crystallization efforts. This chapter presents essential procedures used to generate successful crystals of type V secretion proteins beginning with different options for protein production. Following a description of the preparation and evaluation of crystallization experiments, optimization procedures of initial crystallization conditions are provided. A seeding protocol, employed to grow and obtain larger protein crystals, is also described.