The Mycobacterium tuberculosis (Mtb) FF-ATP synthase (α:β:γ:δ:ε:a:b:b':c) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system α:β:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the α:β:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε, and Mtε, reconstituted with α:β:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.