TRPM7 Ion Channel Structural Determination

At 3.3-Å resolution for the TRPM7-EDTA structure, the cryo-EM density map was of sufficient quality for de novo atomic model building, despite the lower density of the N terminus. For the full-length (1 to 1280) protein, a polyalanine model was first built in COOT (59 (link)). Taking advantage of the defined geometry of helices and clear bumps for Cα atoms in the transmembrane domains, amino acid assignment was subsequently achieved based primarily on the clearly defined side chain densities of bulky residues such as Phe, Tyr, and Trp, as well as some Arg and Lys residues. Resolution of the first part of NT (1 to 150) were insufficient for backbone tracing, and the polyalanine model was used for that region. The refined atomic model was further visualized in COOT. A few residues with side chains moving out of the density during the refinement were fixed manually, followed by further refinement. The TRPM7 model was then subjected to global refinement and minimization in real space using the PHENIX (60 (link)) module “phenix.real_space_refine” (61 ), and geometries of the model were assessed using MolProbity (62 (link)) in the comprehensive model validation section of PHENIX. The final model of TRPM7-EDTA structure exhibited good geometry as indicated by the Ramachandran plot (preferred region, 97.8%; allowed region, 2.1%; outliers, 0.1%). The pore radius was calculated using HOLE (63 (link)). For the TRPM7-DVF and TRPM7-Mg²⁺ structures, the structure of TRPM7-EDTA at 3.3 Å was docked into the cryo-EM maps, followed by manual adjustment in COOT. They were then subjected to the same global refinement and minimization process described for TRPM7-EDTA, and geometries of the models were also assessed using MolProbity (62 (link)). The final models of TRPM7-DVF and TRPM7-Mg²⁺ structures exhibited good geometry as indicated by the Ramachandran plot.