Supplemental Experimental Procedures Purification of TCPH181A Filaments

V. cholerae RT4225 cells were derived from classical V. cholerae O395 and have a mutation in the tcpA gene at the site encoding amino acid 181, resulting in a histidine-to-alanine change (H181A). TCP from RT4225 are shed into the growth media, allowing purification of milligram quantities of pili. RT4225 contains the pMT5 plasmid with an ampicillin resistance marker and an inducible toxT gene that encodes for a transcriptional activator of the tcp operon (DiRita et al., 1996). ToxT over-expression results in increased pilus expression. V. cholerae RT4225 cells were grown for 16 hours under TCP-expressing conditions (Luria broth [LB], pH 6.5, 30 ºC) on a roller shaker in media supplemented with 100 μg/ml ampicillin and 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG). Cultures were centrifuged to remove cells, and additional impurities were removed from the culture supernatant with a 10 % ammonium sulfate precipitation. TCPH181A were precipitated from the supernatant using 30 % ammonium sulfate. The TCPH181A pellet was resuspended in phosphate-buffered saline (PBS) with 10 mM EDTA and dialyzed exhaustively in PBS/EDTA solution to remove ammonium sulfate.