Ystem (T3SS), and form IV pili (9). RsmA negatively controls variables linked with chronic colonizationpnas.org/cgi/doi/10.1073/pnas.Thomologs (RsmA and RsmE) (13, 14), only RsmA had been identified within the opportunistic human pathogen P. aeruginosa (15). A homology search of the P. aeruginosa strain PAO1 genome identified a smaller ORF situated inside the intergenic area between genes PA5183 and PA5184 (SI Appendix, Fig. S1A). The predicted ORF encodes a 71-aa protein bearing 31 identity and 53 similarity to RsmA (Fig. 1A). Provided the restricted homology on the putative gene solution with CsrA, RsmA, and RsmE, the gene was designated rsmF. All previously characterized CsrA proteins possess a extremely conserved secondary structure consisting of five -strands in addition to a carboxyl-terminal (C-terminal) -helix (4, 13, 16, 17). Analysis with the predicted RsmF sequence revealed a one of a kind insertion among -strands 2 and three, in addition to a C-terminal deletion relative to other CsrA family members (Fig. 1A).Author contributions: J.N.M., M.R.D., C.J.G., M.L.U., T.L.Y., and M.C.W. made analysis; J.N.M., M.R.D., W.G.W., C.J.G., L.B., M.L.U., T.L.Y., and M.C.W. performed research; J.N.M., M.R.D., C.J.G., M.L.U., T.L.Y., and M.C.W. contributed new reagents/ analytic tools; J.N.M.Buy1842337-34-1 , M.Fmoc-8-Aoc-OH structure R.PMID:33602077 D., W.G.W., C.J.G., L.B., M.L.U., M.R.R., T.L.Y., and M.C.W. analyzed information; and J.N.M., M.R.D., C.J.G., M.R.R., T.L.Y., and M.C.W. wrote the paper. The authors declare no conflict of interest. This article is usually a PNAS Direct Submission. Data deposition: The RsmF coordinates and structure elements have already been deposited inside the Protein Data Bank, pdb.org (PDB ID code 4K59). The RsmF primary sequence has been deposited in the GenBank database [accession no. KF364633 (strain PA103)].1J.N.M. and M.R.D. contributed equally to this work. To whom correspondence needs to be addressed. E-mail: [email protected]. edu.This article consists of supporting facts online at pnas.org/lookup/suppl/doi:10. 1073/pnas.1307217110/-/DCSupplemental.PNAS | September 10, 2013 | vol. 110 | no. 37 | 15055?MICROBIOLOGYAB13C53341 four 44Fig. 1. RsmF structure. (A) Main sequence alignment of E. coli (Ec) CsrA, P. aeruginosa (Pa) RsmA and RsmF, and P. fluorescens (Pf) RsmA and RsmE. All 5 proteins consist of five -strands (1?) and a single primary -helix (1), but the organization of those components is distinct for RsmF. Conserved arginine residues required for maximal CsrA/RsmA RNA-binding activity are boxed. (B and C) Ribbon diagrams of the RsmF crystal structure as a homodimer (B) and also the reported resolution structure of P. fluorescens dimeric RsmE (pdb ID 2JPP), a homolog of P. aeruginosa RsmA (C).To identify regardless of whether RsmF maintained the general architecture of other CsrA proteins, we determined the crystal structure at two.2-?resolution and refined it to R and Rfree values of 0.21 and 0.27, respectively (SI Appendix, Table S1). RsmF forms a dimer, with residues 1?five of each monomer ordered in the final structure (Fig. 1B). The RsmF dimer is made by two antiparallel -sheets, each composed of 1, three, and 4 from 1 protein monomer, and two and five from the other (SI Appendix, Fig. S2A). The -helices of each and every RsmF monomer, positioned involving -strands 2 and 3, interact with each other and are located above the central region of the dimer (Figs. 1B and SI Appendix, S2A). This arrangement differs from CsrA family members of known structure in that the antiparallel -sheets are composed of 1 and five from a single monomer and 2, 3, and four in the oth.