Objective The objective of this study is to structurally characterize the interaction between the Pseudomonas aeruginosa PA01extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, and the sigma regulator, FpvR. Hypothesis The extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, interacts with high affinity with the N‐terminal anti‐sigma domain of FpvR. It can be purified to homogeneity and used for structural studies by X‐ray crystallography. Summary Transcription of the pyoverdine transport system of Pseudomonas aeruginosa PA01 is mediated by Cell Surface Signaling (CSS). This process involves binding of the siderophore ferric‐pyoverdine to the outer membrane transporter FpvA. This event triggers the regulated intramembrane proteolysis of the sigma regulator, FpvR. Release of the FpvR anti‐sigma domain (ASD) in complex with the extra‐cytoplasmic‐function (ECF) sigma factor FpvI, directs RNA polymerase to the promoter of FpvA to initiate the transcription. The molecular details of the interaction between FpvR ASD and FpvI are not known. We are using a dual expression plasmid to produce the FpvR ASD and a his‐tagged construct of FpvI. Immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography is used to purify the complex. We have characterized the complex by Circular dichroism (CD) to show it is well folded. This protein is being further characterized by sparse matrix crystal screening and Small‐angle X‐ray scattering (SAXS).