Fig. 2: The closed permeation pathway and Mg²⁺-binding sites.

One selected monomer is shown in orange throughout. a, MOLE software analysis of the conducting pore, shown as electrostatic surface, with residues lining the pathway shown as sticks. b, HOLE software calculation of pore diameter along the pore. c, Location of putative Mg²⁺ ions (shown in green) in the closed homopentamer. d, Close-up views with supporting cryo-EM density in the symmetry-applied C5 map of the putative Mg²⁺-binding sites close to or in the conducting pathway. Site U is positioned next to the loop in between TM1 and TM2 and site S is positioned next to the GMN motif of the selectivity filter. Sites P1 and P2 are located in the TM domain. e, Close-up views of sites M1–M4 in the NTD of CtMrs2. f, Close-up views of the corresponding Mg²⁺-binding sites (shown in e) in hMrs2 (PDB 8IP3), equivalent to sites M1 (top) and M4 (bottom). g, Growth phenotypes on YPD and YPG (the latter requiring mitochondrial respiration) media of WT Mrs2 from S. cerevisiae (ScMrs2), the equivalent MRS2-knockout strain (−MRS2), and cells based on −MRS2 with an empty vector or a vector containing different mutant forms of ScMrs2 (CtMrs2 numbering).