Is MGSSHHHHHHSSGLVPR/GSH in a single letter amino acid code (thrombin recognition sequence and cleavage site indicated by an underline and `/’, respectively). Cells were grown to the OD600nm of 1.2 at 37 in Super Broth and were induced to express the fusion protein with 1mM IPTG for 15 hrs at 18 . Cells were centrifuged at 4,700 g and the cell pellets were resuspended in TBS (20 mM Tris pH 8.0, 150 mM NaCl) buffer containing 10 mM imidazole. The cells were treated with lysozyme, freeze-thawed and lysed by sonication. The protein was purified with Ni-NTA metal affinity chromatography, followed by N-terminal his tag removal by thrombin cleavage. The tag-less GFP-Bak was purified by another round of Ni-NTA metal affinity chromatography in TBSMethodsScientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/Figure 5. Schematic model of a lipidic pore formed by Bak homodimers. (a) The BGHs line the lumen of the lipidic pore. Helices 6-8 are adsorbed to the toroidal surface of the pore and anchored to the transmembrane helix 9 that stays in the flat region of the membrane around the pore. The three separate oligomerization interfaces are indicated. Numbered rectangles are the EPZ004777 site corresponding helices in Bak (Helices 6-9 were omitted for two gray-colored Bak molecules and helix 1 omitted for all for clarity.). Of note, the BGHs may exist close to the flat region/toroidal surface boundary of the lipidic pore. (b) Two possible arrangements of 5-6 domains are shown in a cross-sectional side view of a schematic Bak lipidic pore. These correspond to the two possible orientations of 5 helices (in cyan) in a BGH that is adsorbed onto the toroidal surface (See the two 5-6 domains in two separate BGHs shown in blue and cyan in (a)).buffer. The protein was concentrated to a final concentration of 20 mg/ml using Amicon Ultra concentrator with the molecular weight cutoff of 50 kDa (Millipore). Crystallization of GFP-Bak, data collection and structure determination. Purified GFP-Bak was crystallized by hanging-drop vapor diffusion method adapting Czabotar et al.25 as described in the Supplementary Information. Diffraction data were collected at the GM/CA-CAT, Advanced Photon Sources in Argonne National Laboratory and the structure was determined by molecular replacement as described in detail in the Supplementary Information (also see Table 1).Chemical cross-linking experiments. Cell culture. The wild type and bax-/- bak-/- double knockout mouse embryonic fibroblasts (MEFs)45 were obtained from the laboratory of Stanley Korsmeyer (Dana-Farber Cancer Institute, Boston, MA). MEFs were cultured on cell culture dishes (Genesee, San Diego, CA) in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10 heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 M nonessential amino acids (NEAA), 1 penicillin/streptomycin at 37 in a 5 CO2 incubator.Scientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/Preparation of MEF cell lines expressing mutant Bak protein. Retroviral plasmids harboring genes for single, double, or triple cysteine substitution BAK mutant proteins were prepared with the template plasmid pMSCV-mBAK-IRES-GFP46 using the SB 202190 supplier QuikChange Site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Retroviral preparations were made by cotransfecting the ecotropic 293 cells with pMSCV-mBAK-IRES-GFP vector, pECO and pSG5-BCLx using Lipofe.Is MGSSHHHHHHSSGLVPR/GSH in a single letter amino acid code (thrombin recognition sequence and cleavage site indicated by an underline and `/’, respectively). Cells were grown to the OD600nm of 1.2 at 37 in Super Broth and were induced to express the fusion protein with 1mM IPTG for 15 hrs at 18 . Cells were centrifuged at 4,700 g and the cell pellets were resuspended in TBS (20 mM Tris pH 8.0, 150 mM NaCl) buffer containing 10 mM imidazole. The cells were treated with lysozyme, freeze-thawed and lysed by sonication. The protein was purified with Ni-NTA metal affinity chromatography, followed by N-terminal his tag removal by thrombin cleavage. The tag-less GFP-Bak was purified by another round of Ni-NTA metal affinity chromatography in TBSMethodsScientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/Figure 5. Schematic model of a lipidic pore formed by Bak homodimers. (a) The BGHs line the lumen of the lipidic pore. Helices 6-8 are adsorbed to the toroidal surface of the pore and anchored to the transmembrane helix 9 that stays in the flat region of the membrane around the pore. The three separate oligomerization interfaces are indicated. Numbered rectangles are the corresponding helices in Bak (Helices 6-9 were omitted for two gray-colored Bak molecules and helix 1 omitted for all for clarity.). Of note, the BGHs may exist close to the flat region/toroidal surface boundary of the lipidic pore. (b) Two possible arrangements of 5-6 domains are shown in a cross-sectional side view of a schematic Bak lipidic pore. These correspond to the two possible orientations of 5 helices (in cyan) in a BGH that is adsorbed onto the toroidal surface (See the two 5-6 domains in two separate BGHs shown in blue and cyan in (a)).buffer. The protein was concentrated to a final concentration of 20 mg/ml using Amicon Ultra concentrator with the molecular weight cutoff of 50 kDa (Millipore). Crystallization of GFP-Bak, data collection and structure determination. Purified GFP-Bak was crystallized by hanging-drop vapor diffusion method adapting Czabotar et al.25 as described in the Supplementary Information. Diffraction data were collected at the GM/CA-CAT, Advanced Photon Sources in Argonne National Laboratory and the structure was determined by molecular replacement as described in detail in the Supplementary Information (also see Table 1).Chemical cross-linking experiments. Cell culture. The wild type and bax-/- bak-/- double knockout mouse embryonic fibroblasts (MEFs)45 were obtained from the laboratory of Stanley Korsmeyer (Dana-Farber Cancer Institute, Boston, MA). MEFs were cultured on cell culture dishes (Genesee, San Diego, CA) in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10 heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 M nonessential amino acids (NEAA), 1 penicillin/streptomycin at 37 in a 5 CO2 incubator.Scientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/Preparation of MEF cell lines expressing mutant Bak protein. Retroviral plasmids harboring genes for single, double, or triple cysteine substitution BAK mutant proteins were prepared with the template plasmid pMSCV-mBAK-IRES-GFP46 using the QuikChange Site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Retroviral preparations were made by cotransfecting the ecotropic 293 cells with pMSCV-mBAK-IRES-GFP vector, pECO and pSG5-BCLx using Lipofe.
