Abstract
We report the cryoEM structure of the Francisella protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the Francisella genus, which we now designate FPM13 (Francisella Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of Francisella type VI secretion system (T6SS) effector proteins, co-purifying with them. Its identity, initially unknown, was established using the novel cryoID method. The structure reveals a symmetrical, donut-shaped 18-mer with 9-fold dihedral symmetry, formed by two stacked nonamers head-to-head. It measures ~8 nm both in height and in outer diameter, and has a 3.5 nm central channel. The complex features a double-layered wall with an inner β-sheet core and an outer α-helical shell. Each monomer adopts a compact fold comprising an N-terminus β-strand, an α-helix and two additional β strands at the C-terminus. The assembly is stabilized by inter-ring loop interactions and hydrophobic and electrostatic contacts between neighboring subunits. Biochemical analyses, as shown by APEX-biotinylation and Triton X-114 phase partitioning, confirmed that FPM13 is a soluble periplasmic protein. ICP-MS demonstrated that FPM13 binds iron and copper. Deletion of FPM13 in Francisella novicida strains caused no growth defects in macrophages or mice but show increased copper sensitivity under iron-depleted conditions, suggesting a role for FPM13 in metal transport or detoxification.