Fig. 4: 3D-LAFM steered MDFF structural models of Glt_Ph IFS states.

a PDB structure of Glt_Ph IFS (PDB 6X12). A Glt_Ph trimer consists of three functionally independent transport domains (green) and a central trimerization domain (blue). Top: Glt_Ph trimer viewed from the cytoplasmic side (left) and Glt_Ph monomer displayed in side view (right). Bottom: Cartoon representation of Glt_Ph monomer, where transport domain tilt change with respect to the z-axis is a characteristic distinguishing IFS_open (left) and IFS_closed (right) conformations. b Structures (left, surface representation from the cytoplasmic side) of IFS_open (top, PDB 6X12) and IFS_closed (bottom, PDB 4P19) Glt_Ph, and high-density 3D-LAFM surfaces (right). 3D-LAFM data were modified from ref. ¹⁰. c CVs tracked during MDFF: Top left: angle between transport domain and z-axis (angle θ). Bottom left: Height difference between transport and trimerization domains on the cytoplasmic side (Δz). Right: Angle between trimerization domain and z-axis (angle φ). d–i Analyses of ‘trans-fitting’ 3D-LAFM MDFF trajectories: d, g: U_AFM–IFSo used to steer IFSc-apo-X-ray. e, h: U_AFM–IFSo used to steer IFSc-trsp-EM, and f, i: U_AFM–IFSc used to steer IFSo-apo-EM (Methods). d–f Gray: U_AFM force fields derived from 3D-LAFM density maps. Scarlet: MDFF structural models displayed from the cytoplasmic side (top) and in a side view (bottom). Left: Initial model (t = 0 ns). Right: Final model (t ~ 60 ns). g–i 1st, 2nd, and 3rd panel: CVs φ, θ, Δz, where the gray lines represent the evolution of the three individual transporter domains (protomers 1, 2, and 3). Reference structures: IFSo-apo-EM (red dashed line), IFSc-apo-X-ray (black dashed line), and IFSc-trsp-EM (blue dashed line). 4th panel: rmsd of the MDFF structures to the initial PDB structure (black) and the ‘target’ structure (gray). The ‘cis-fitting’ 3D-LAFM MDFF trajectories are reported in Supplementary Fig. 5.