Lactose permease of E.coli.(LacY)is a secondary active transporter(SAT)that belongs to the major facilitator superfamily(MFS).Experimental structures of the cytoplasmic-open and more recently occluded-like structure have been determined,however,the crystal structure of LacY in the periplasmic-open state is still not available,while it is important for understanding complete proton/sugar transport process of LacY as well as other similar SAT proteins.Previously,a structural model of periplasmic-open LacY has been obtained through a two-step hybrid implicit-explicit(IMEX)simulation method [1].Since DEER is the only successful experimental method to date that provides detail of the outward-facing LacY conformation by measuring the distance of spin labels attached to residues,molecular dynamics dummy spin-label(MDDS)simulations are performed on all three states of LacY to test the effect of spin labels(size and internal flexibility)on DEER spin label distance measurements.The results show that the orientation and movement of the spin labels significantly affect the residue pair distance.Self-guided Langevin dynamics(SGLD)[2] simulations can be used to obtain LacY structures of all the conformational states efficiently by enhancing the conformational sampling of low frequency motions.SGLD simulations are performed on preequilibrated(1PV7)inward-facing,occluded,and unphysical open-open(i.e.both peri-and cytoplasmic open)states of LacY with substrate ββ-(Galp)2 with varied guiding factor(sgft),friction coefficients(fbeta),by applying guiding force on entire protein and partially(C-domain and Ndomain,periplasmic-side,and LacY-substrate binding pocket).The simulation results of pore radii and residue pair distance change for these cases will be presented.To obtain a potentially more accurate periplasmic-open LacY structure,MD simulations with the explicit membrane are performed on the(partially)periplasmic-open structure obtained from SGLD simulations(i.e.IM-EX simulation method),and the simulation results will also be presented.