( ) Rfree ( ) Mean B (?) Root-mean-squared deviations Bonds (? Angles (deg) Ramachandran plot statistics

( ) Rfree ( ) Mean B (?) Root-mean-squared deviations Bonds (? Angles (deg) Ramachandran plot statistics ( ) Most favored Additional allowed Generously allowed Disallowed 89.9 9.7 0.4 0.0 0.018 2.14 13.1 0.8 (2.9?.8) 24.36 27.63 87.16 15.1 77.7 (2.9?.8) 97.4 81.7 (2.9?.8) 50?.8 P3121 171.64, 171.64, 98.19 90, 90, 120 1.0 GM/CA-CAT, APS 584081Table 1. Statistics of the X-ray diffraction data. Rsym = j i|Iij – | / i j Iij, where i runs over multiple observations of the same intensity, and j runs over all crystallographic unique intensities. Rfactor = ||Fobs| – |Fcalc||/|Fobs|. Rfree was calculated with 5 of the reflections selected.was brought close to each other between BGHs in the Bak oligomeric pore, but not within a single BGH since the two 143C residues in a Bak BGH (i.e., 143C and 143C in Fig. 2a) are separated too far away for disulfide formation ( 50 ?between C atoms). In conclusion, the above results showed that the C-termini of 5 helices around residue 143 in the BGHs were near the Bak oligomerization interface. Likewise, even-numbered high order oligomers were formed in a p7/p15 Bid-dependent manner only in Bak 69C/111C/96C but not in Bak 96C (Fig. 2g; lanes 5 and 6, and lanes 1 and 2, respectively). The large distance between two 96C residues in a BGH ( 45 ?between C atoms) also GW 4064 molecular weight precludes the possibility of disulfide bond formation within a BGH. Thus, these results indicated that residue 96C, i.e., the C-termini of 3 helices, were juxtaposed in the oligomeric Bak between neighboring BGHs. Finally, similar results were also observed in Bak 162C and Bak 69C/111C/162C (lanes 3, 4, 7 and 8), indicating that residue 162C, the penultimate C-terminal residue of helix 6, was also at the oligomerization interface in Bak pore, consistent with the formerly known `6:6 interface’23. Additionally, the monomers of Bak 86C were cross-linked upon activation by p7/p15 Bid, consistent with the proximity of the two symmetry-related 86C residues in the BGH structure (86C and 86C; 10 ?or 13 ?between C or C atoms, respectively) (Fig. 2h). This also indicated that the BGH structure was preserved in the Bak oligomeric pores in membrane. The above results collectively showed that, in addition to 6 helices, the C-termini of helices 3 and 5 were juxtaposed between BGHs in oligomeric Bak (Fig. 2a) in apoptotic mitochondria, thus demonstrating the existence of the `3/5 interface.’ This is consistent with our earlier in vitro results obtained with recombinant mutant Bak proteins in liposomes27. Using site-directed spin labeling (SDSL) method (Fig. 3a), the inter-spin distances in the range of 15-80 ?can be measured by the double electron electron resonance (DEER) method36. There would be multiple spin-spin interactions between BGHs as well as within a BGH if spin-labeled Bak monomers formed oligomeric Bak pores (Fig. 3b,c). This was indeed the case in the Bak oligomeric pores formed with Bak/84R1, a Bak monomer spin labeled at residue 84 (Fig. 3d,e; also see Supplementary Information Figure S2). Clearly, three well-resolved peaks were observed in the probability vs. distance function obtained from the X-band DEER data using BFA price DeerAnalysis2013 program37 (Fig. 3d). Due to the short phase memory time of the electronic spins of the nitroxide labels in X-band experiment, the evolution time was limited and thus the accuracy of the longer distance was compromised. The Q-band DEER was thus used to overcome this. As shown in Fig. 3e (left panel), the evolution.( ) Rfree ( ) Mean B (?) Root-mean-squared deviations Bonds (? Angles (deg) Ramachandran plot statistics ( ) Most favored Additional allowed Generously allowed Disallowed 89.9 9.7 0.4 0.0 0.018 2.14 13.1 0.8 (2.9?.8) 24.36 27.63 87.16 15.1 77.7 (2.9?.8) 97.4 81.7 (2.9?.8) 50?.8 P3121 171.64, 171.64, 98.19 90, 90, 120 1.0 GM/CA-CAT, APS 584081Table 1. Statistics of the X-ray diffraction data. Rsym = j i|Iij – | / i j Iij, where i runs over multiple observations of the same intensity, and j runs over all crystallographic unique intensities. Rfactor = ||Fobs| – |Fcalc||/|Fobs|. Rfree was calculated with 5 of the reflections selected.was brought close to each other between BGHs in the Bak oligomeric pore, but not within a single BGH since the two 143C residues in a Bak BGH (i.e., 143C and 143C in Fig. 2a) are separated too far away for disulfide formation ( 50 ?between C atoms). In conclusion, the above results showed that the C-termini of 5 helices around residue 143 in the BGHs were near the Bak oligomerization interface. Likewise, even-numbered high order oligomers were formed in a p7/p15 Bid-dependent manner only in Bak 69C/111C/96C but not in Bak 96C (Fig. 2g; lanes 5 and 6, and lanes 1 and 2, respectively). The large distance between two 96C residues in a BGH ( 45 ?between C atoms) also precludes the possibility of disulfide bond formation within a BGH. Thus, these results indicated that residue 96C, i.e., the C-termini of 3 helices, were juxtaposed in the oligomeric Bak between neighboring BGHs. Finally, similar results were also observed in Bak 162C and Bak 69C/111C/162C (lanes 3, 4, 7 and 8), indicating that residue 162C, the penultimate C-terminal residue of helix 6, was also at the oligomerization interface in Bak pore, consistent with the formerly known `6:6 interface’23. Additionally, the monomers of Bak 86C were cross-linked upon activation by p7/p15 Bid, consistent with the proximity of the two symmetry-related 86C residues in the BGH structure (86C and 86C; 10 ?or 13 ?between C or C atoms, respectively) (Fig. 2h). This also indicated that the BGH structure was preserved in the Bak oligomeric pores in membrane. The above results collectively showed that, in addition to 6 helices, the C-termini of helices 3 and 5 were juxtaposed between BGHs in oligomeric Bak (Fig. 2a) in apoptotic mitochondria, thus demonstrating the existence of the `3/5 interface.’ This is consistent with our earlier in vitro results obtained with recombinant mutant Bak proteins in liposomes27. Using site-directed spin labeling (SDSL) method (Fig. 3a), the inter-spin distances in the range of 15-80 ?can be measured by the double electron electron resonance (DEER) method36. There would be multiple spin-spin interactions between BGHs as well as within a BGH if spin-labeled Bak monomers formed oligomeric Bak pores (Fig. 3b,c). This was indeed the case in the Bak oligomeric pores formed with Bak/84R1, a Bak monomer spin labeled at residue 84 (Fig. 3d,e; also see Supplementary Information Figure S2). Clearly, three well-resolved peaks were observed in the probability vs. distance function obtained from the X-band DEER data using DeerAnalysis2013 program37 (Fig. 3d). Due to the short phase memory time of the electronic spins of the nitroxide labels in X-band experiment, the evolution time was limited and thus the accuracy of the longer distance was compromised. The Q-band DEER was thus used to overcome this. As shown in Fig. 3e (left panel), the evolution.