Discovery and CryoEM Structure of FPM13, a Periplasmic Metalloprotein Unique to .

We report the cryoEM structure of the protein FTN_1118, identifying it as a novel 13 kDa periplasmic protein unique to the genus, which we now designate FPM13 ( Periplasmic Metalloprotein, 13 kDa) based on its structural and biochemical properties. FPM13 was serendipitously identified during purification of 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 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.