M2 protein of influenza A virus forms a homo-tetrameric proton channel involved in modifying virion and trans-Golgi pH during virus infection and inhibited by drugs Amantadine and Rimantadine. Previously determined structures for the membrane domain of M2 protein in detergent micelles have shown different conformations of TM helices with respect to channel axis i.e. tilt as well as relative orientation than structures solved in lipid bilayer environment. We report a new three dimensional structure of closed channel state of M2 protein residue (22-62) in native like PC:PE bilayer that encompasses all functionally relevant domains for proton channel activity.In order to obtain high resolution structure in bilayer environment, M2(22-62) was expressed and purified from E. Coli. membrane and reconstituted in liposomes. Liposomal assays demonstrated fully active and amantadine-sensitive channels with an average proton uptake of 21.1±1.9 H+/tetramer/s. Multidimensional Solid State NMR experiments performed on uniform 15N labeled and amino acid specific labeled M2(22-62) reconstituted in a DOPC:DOPE(4:1) lipid bilayer generated precise orientational restraints for amide bond vectors and peptide planes for each residue to determine three-dimensional structure. Helical tilt and rotation were calculated using dipolar couplings, chemical shift wave and polarization inversion slant angle (PISA) wheel analysis. Tetramer assembly and membrane equilibration was performed using molecular dynamics simulations. Structure shows four-helix bundle with TM and amphipathic helices tilted at ∼34 degrees and ∼105 degrees to channel axis, respectively with a tight turn joining two helices. Although the oligomeric state of the channel is stabilized due to the interactions of amphipathic helices as previously reported, membrane interaction and rotational orientation of amphipathic helices is significantly different with bulky hydrophobic residues buried deep in the membrane. Salient features of the structure will be reported in light of proton transport mechanism.