Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli CusB

This article describes how CusB from E. coli was overexpressed and the recombinant protein purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography, and how the purified CusB protein was crystallized using the vapour-diffusion method. Periplasmic membrane-fusion proteins (MFPs) are an essential component of multidrug and metal-efflux pumps in Gram-negative bacteria. However, the functional structure of MFPs remains unclear. CusCFBA, the Cu^I and Ag^I efflux system in Escherichia coli, consists of the MFP CusB, the OMF CusC and the RND-type transporter CusA. The MFP CusB bridges the inner membrane RND-type efflux transporter CusA and the outer membrane factor CusC and exhibits substrate-linked conformational changes which distinguish it from other MFP-family members. CusB from E. coli was overexpressed and the recombinant protein was purified using Ni–NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified CusB protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to a resolution of 3.1 Å at 100 K. The crystal belonged to space group C222