With each other, forming the `9:9 interface’ within the flat region of the membrane around the lipidic pore as suggested by others28,31,40. The extended 6-8 helices, while being adsorbed to the lipidic pore lumen, are hypothesized to be in a pseudo-parallel orientation27, tethering the BGHs to 9 helices. This forms the `6:6 interfaces’ between the neighboring Bak homodimers, consistent with our and others’ results23,27,41. In this model, the topological arrangements of 5-6 helical hairpins on the lipidic pore surface can also be explained as schematically shown in Fig. 5b. When a BGH is adsorbed within the curved surface of the lipidic pore as depicted in Fig. 5a, the 5-6 helical segment is inserted into the membrane with their helical axes tilted toward the C- and N-terminus, respectively (Fig. 5b), consistent with the experimental results (Fig. 4d). Note that the two possible arrangements of the 5-6 helical hairpins in Fig. 5b correspond to the two conformations of each monomer in the BGH adsorbed to the lipidic pore (Fig. 5a). Also note that the C-termini of 3 and 5 helices can be brought to a close proximity between BGHs of any neighboring Bak homodimers, which are presumably on the curved surface of the lipidic pore, forming the `3/5 interface’ (Fig. 5a). Currently, the exact location of BGHs within the lipidic pore is not known18,19,21. The dimension of a BGH is approximately 40 ?from top to bottom when oriented as depicted in Fig. 5a, which can be placed within the lipidic pore. The topology of Bax pores or arcs recently determined by the atomic force microscopy (AFM)18 showed a protrusion of about 40 (?0) ?at the rim relative to the membrane surface. Earlier, Epand et al. buy SB 202190 reported a protrusion of 24 ?at the pore rim in an AFM image of Bax pore with the diameter of 20 nm42. These suggest that BGHs and/or other domains (e.g., helices 1 or 6-9) of Bax or Bak might cover both the flat region and the toroidal surface of the lipidic pore. Finally, noting that a simple 2-dimensional triangulation with the measured intra- and inter-dimer distances could explain the proximity of residues in the `3/5 interface’ (Fig. 3), the diameters of the lipidic pores formed by Bak in liposomes in this study should be very large, which is consistent with other’s reports18?1,42,43. In conclusion, we have determined the mouse Bak BGH structure, which allowed more accurate modeling of the DEER distances observed within and between the BGHs. The curved hydrophobic surface of the BGH was immersed in the membrane at a shallow depth. The BGHs were shown to oligomerize via the `3/5 interface’ in mitochondria. These findings led us to propose a probable assembly of the Bak homodimers in the mitochondrial apoptotic pore. This sheds important insights into the action mechanism of Bak or Bax in mitochondrial apoptosis pathway. However, the function of the novel `3/5 interface’ in Bak oligomerization and pore formation is unknown and it requires further investigation.X-ray crystallography. Protein expression and purification. A DNA fragment for 2-5 of mouse BAK (residues 66-144) was subcloned into the pET-28a-GFP A206N vector (designated as pYEGFP_A206N _BAK_ H2-H5_pET28a)25,44. The resulting vector expresses an amino (N)-MS023 supplement terminally hexahistidine tagged green fluorescent protein (with A206N mutation) fused to mouse Bak 2-5 helices (designated as His-GFP-Bak) in E. coli BL21 (DE3) (see Fig. 1a). The N-terminal sequence of His-tag and the thrombin cleavage sequence.With each other, forming the `9:9 interface’ within the flat region of the membrane around the lipidic pore as suggested by others28,31,40. The extended 6-8 helices, while being adsorbed to the lipidic pore lumen, are hypothesized to be in a pseudo-parallel orientation27, tethering the BGHs to 9 helices. This forms the `6:6 interfaces’ between the neighboring Bak homodimers, consistent with our and others’ results23,27,41. In this model, the topological arrangements of 5-6 helical hairpins on the lipidic pore surface can also be explained as schematically shown in Fig. 5b. When a BGH is adsorbed within the curved surface of the lipidic pore as depicted in Fig. 5a, the 5-6 helical segment is inserted into the membrane with their helical axes tilted toward the C- and N-terminus, respectively (Fig. 5b), consistent with the experimental results (Fig. 4d). Note that the two possible arrangements of the 5-6 helical hairpins in Fig. 5b correspond to the two conformations of each monomer in the BGH adsorbed to the lipidic pore (Fig. 5a). Also note that the C-termini of 3 and 5 helices can be brought to a close proximity between BGHs of any neighboring Bak homodimers, which are presumably on the curved surface of the lipidic pore, forming the `3/5 interface’ (Fig. 5a). Currently, the exact location of BGHs within the lipidic pore is not known18,19,21. The dimension of a BGH is approximately 40 ?from top to bottom when oriented as depicted in Fig. 5a, which can be placed within the lipidic pore. The topology of Bax pores or arcs recently determined by the atomic force microscopy (AFM)18 showed a protrusion of about 40 (?0) ?at the rim relative to the membrane surface. Earlier, Epand et al. reported a protrusion of 24 ?at the pore rim in an AFM image of Bax pore with the diameter of 20 nm42. These suggest that BGHs and/or other domains (e.g., helices 1 or 6-9) of Bax or Bak might cover both the flat region and the toroidal surface of the lipidic pore. Finally, noting that a simple 2-dimensional triangulation with the measured intra- and inter-dimer distances could explain the proximity of residues in the `3/5 interface’ (Fig. 3), the diameters of the lipidic pores formed by Bak in liposomes in this study should be very large, which is consistent with other’s reports18?1,42,43. In conclusion, we have determined the mouse Bak BGH structure, which allowed more accurate modeling of the DEER distances observed within and between the BGHs. The curved hydrophobic surface of the BGH was immersed in the membrane at a shallow depth. The BGHs were shown to oligomerize via the `3/5 interface’ in mitochondria. These findings led us to propose a probable assembly of the Bak homodimers in the mitochondrial apoptotic pore. This sheds important insights into the action mechanism of Bak or Bax in mitochondrial apoptosis pathway. However, the function of the novel `3/5 interface’ in Bak oligomerization and pore formation is unknown and it requires further investigation.X-ray crystallography. Protein expression and purification. A DNA fragment for 2-5 of mouse BAK (residues 66-144) was subcloned into the pET-28a-GFP A206N vector (designated as pYEGFP_A206N _BAK_ H2-H5_pET28a)25,44. The resulting vector expresses an amino (N)-terminally hexahistidine tagged green fluorescent protein (with A206N mutation) fused to mouse Bak 2-5 helices (designated as His-GFP-Bak) in E. coli BL21 (DE3) (see Fig. 1a). The N-terminal sequence of His-tag and the thrombin cleavage sequence.