1bcc. Lichtarge lab Evolutionary trace report by report maker May 29, 2010 CONTENTS

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1 Pages bcc Evolutionary trace report by report maker May 29, Chain 1bccJ P00130 overview Multiple sequence alignment for 1bccJ Residue ranking in 1bccJ Top ranking residues in 1bccJ and their position on the structure Clustering of residues at 25% coverage Overlap with known functional surfaces at 25% coverage Possible novel functional surfaces at 25% coverage. 18 CONTENTS 1 Introduction 1 2 Chain 1bccF P00129 overview Multiple sequence alignment for 1bccF Residue ranking in 1bccF Top ranking residues in 1bccF and their position on the structure Clustering of residues at 25% coverage Overlap with known functional surfaces at 25% coverage Possible novel functional surfaces at 25% coverage. 5 3 Chain 1bccA P31800 overview Multiple sequence alignment for 1bccA Residue ranking in 1bccA Top ranking residues in 1bccA and their position on the structure Clustering of residues at 25% coverage Overlap with known functional surfaces at 25% coverage Possible novel functional surfaces at 25% coverage Chain 1bccE P13272 overview Multiple sequence alignment for 1bccE Residue ranking in 1bccE Top ranking residues in 1bccE and their position on the structure Clustering of residues at 25% coverage Overlap with known functional surfaces at 25% coverage Possible novel functional surfaces at 25% coverage Chain 1bccB P23004 overview Multiple sequence alignment for 1bccB Residue ranking in 1bccB Top ranking residues in 1bccB and their position on the structure Clustering of residues at 23% coverage Overlap with known functional surfaces at 23% coverage Possible novel functional surfaces at 23% coverage Chain 1bccH P00126 overview Multiple sequence alignment for 1bccH Residue ranking in 1bccH Top ranking residues in 1bccH and their position on the structure Clustering of residues at 24% coverage Overlap with known functional surfaces at 24% coverage Possible novel functional surfaces at 24% coverage Chain 1bccC Q7GTU5 overview 31 1 Lichtarge lab 2006

2 8.2 Multiple sequence alignment for 1bccC Residue ranking in 1bccC Top ranking residues in 1bccC and their position on the structure Clustering of residues at 28% coverage Overlap with known functional surfaces at 28% coverage Possible novel functional surfaces at 28% coverage Chain 1bccD P00125 overview Multiple sequence alignment for 1bccD Residue ranking in 1bccD Top ranking residues in 1bccD and their position on the structure Clustering of residues at 25% coverage Overlap with known functional surfaces at 25% coverage Possible novel functional surfaces at 25% coverage Chain 1bccG P13271 overview Multiple sequence alignment for 1bccG Residue ranking in 1bccG Top ranking residues in 1bccG and their position on the structure Clustering of residues at 26% coverage Overlap with known functional surfaces at 26% coverage Possible novel functional surfaces at 26% coverage Notes on using trace results Coverage Known substitutions Surface Number of contacts Annotation Mutation suggestions Appendix File formats Color schemes used Credits Alistat CE DSSP HSSP LaTex Muscle Pymol Note about ET Viewer Citing this work About report maker Attachments 58 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1bcc): Title: Cytochrome bc1 complex from chicken Compound: Mol id: 1; molecule: ubiquinol cytochrome c oxidoreductase; chain: a; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 2; molecule: ubiquinol cytochrome c oxidoreductase; chain: b; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 3; molecule: ubiquinol cytochrome c oxidoreductase; chain: c; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 4; molecule: ubiquinol cytochrome c oxidoreductase; chain: d; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 5; molecule: ubiquinol cytochrome c oxidoreductase; chain: e; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 6; molecule: ubiquinol cytochrome c oxidoreductase; chain: f; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 7; molecule: ubiquinol cytochrome c oxidoreductase; chain: g; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 8; molecule: ubiquinol cytochrome c oxidoreductase; chain: h; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 9; molecule: ubiquinol cytochrome c oxidoreductase; chain: i; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids; mol id: 10; molecule: ubiquinol cytochrome c oxidoreductase; chain: j; synonym: cytochrome bc1 complex, complex iii; ec: ; other details: model includes ubiquinone and phospholipids Organism, scientific name: Gallus Gallus; 1bcc contains unique chains 1bccF (100 residues), 1bccA (442 residues), 1bccJ (59 residues), 1bccE (196 residues), 1bccB (406 residues), 1bccH (66 residues), 1bccC (379 residues), 1bccD (241 residues), and 1bccG (78 residues) Not enough homologous sequences could be found to permit analysis for chain 1bccI. 2 CHAIN 1BCCF 2.1 P00129 overview From SwissProt, id P00129, 85% identical to 1bccF: Description: Ubiquinol-cytochrome c reductase complex 14 kda protein (EC ) (Complex III subunit VI). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This component is involved in redox-linked proton pumping. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. 2

3 residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 1. Residues in 1bccF colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Subcellular location: Mitochondrial inner membrane. Similarity: Belongs to the UQCRB/QCR7 family. Caution: Was originally thought to be the ubiquinone-binding protein (QP-C). About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 2.2 Multiple sequence alignment for 1bccF For the chain 1bccF, the alignment 1bccF.msf (attached) with 71 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccF.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 71 Total number of residues: 6685 Smallest: 78 Largest: 100 Average length: 94.2 Alignment length: 100 Average identity: 40% Most related pair: 99% Most unrelated pair: 17% Most distant seq: 50% Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 56% eukaryotic ( 11% vertebrata, 7% arthropoda, 21% fungi, 2% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccF.descr. 2.3 Residue ranking in 1bccF The 1bccF sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1bccF can be found in the file called 1bccF.ranks sorted in the attachment. 2.4 Top ranking residues in 1bccF and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein. Figure 2 shows residues in 1bccF colored by their importance: bright red and yellow indicate more conserved/important Fig. 2. Residues in 1bccF, colored by their relative importance. Clockwise: front, back, top and bottom views Clustering of residues at 25% coverage. Fig. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed in Table 1. Table 1. cluster size member color residues red 23 26,27,30,31,34,35,36,46,49 50,51,56,58,61,64,65,75,89 90,92,100,103,107 blue 2 84,86 Table 1. Clusters of top ranking residues in 1bccF Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccD.Table 2 lists the top 25% of residues at the interface with 1bccD. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section Table R T(8) / Q(4) 3

4 Fig. 3. Residues in 1bccF, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 2. continued R(84) K(1) N(1) 56 D Y(39) / N(12) V(2) I(2) D(30) T(5) E(5) Table 2. The top 25% of residues in 1bccF at the interface with 1bccD. Table R (TY)(D)(FW)(VA) 56 D (R)(H)(FW)(K) Table 3. List of disruptive for the top 25% of residues in 1bccF, that are at the interface with 1bccD. Fig. 4. Residues in 1bccF, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccF.) Figure 4 shows residues in 1bccF colored by their importance, at the interface with 1bccD. Interface with 1bccC.Table 4 lists the top 25% of residues at the interface with 1bccC. The following table (Table 5) suggests possible disruptive replacements for these residues (see Section Table D D(98) / L(1) 31 L L(92) / C(2) Y(1) F(1) R(1) 65 A A(85) / G(9) T(4) 34 D N(5) / D(85) H(8) 26 F Y(43) / F(25) L(22) H(1).(2) G(2) S(1) 4

5 Table 4. continued 36 T L(61) / T(18) M(4) C(9) V(1) W(2) I(1) Table 4. The top 25% of residues in 1bccF at the interface with 1bccC. Table D (R)(H)(FYW)(KCG) 31 L (R)(YE)(K)(T) 65 A (KR)(E)(QH)(Y) 34 D (R)(FW)(VCAHG)(Y) 26 F (K)(E)(Q)(D) 36 T (R)(K)(H)(Q) Table 5. List of disruptive for the top 25% of residues in 1bccF, that are at the interface with 1bccC. Figure 5 shows residues in 1bccF colored by their importance, at the interface with 1bccC. Interface with 1bccG.Table 6 lists the top 25% of residues at the interface with 1bccG. The following table (Table 7) suggests possible disruptive replacements for these residues (see Section Table D Y(39) / N(12) V(2) I(2) D(30) T(5) E(5) Table 6. The top 25% of residues in 1bccF at the interface with 1bccG. Table D (R)(H)(FW)(K) Table 7. List of disruptive for the top 25% of residues in 1bccF, that are at the interface with 1bccG. Figure 6 shows residues in 1bccF colored by their importance, at the interface with 1bccG Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bccF surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 7. The residues belonging to this surface patch are listed in Table 8, while Table 9 suggests possible disruptive replacements for these residues (see Section Fig. 5. Residues in 1bccF, at the interface with 1bccC, colored by their relative importance. 1bccC is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccF.) Table 8. res type substitutions(%) cvg 61 R R(100) L L(97)A(2) D D(98)L(1) Y Y(97)S(1)I(1) L L(92)C(2)Y(1) 0.07 F(1)R(1) 30 G G(97)S(1).(1) A A(85)G(9)T(4) R R(91)G(2)H(4) 0.10 M(1) 34 D N(5)D(85)H(8)

6 Fig. 6. Residues in 1bccF, at the interface with 1bccG, colored by their relative importance. 1bccG is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccF.) Table 8. continued res type substitutions(%) cvg A(1)Y(1).(1) 64 R T(8)Q(4)R(84) 0.15 K(1)N(1) 92 P P(85)D(9)E(2) 0.16 L(1) 51 P P(85)S(2)Q(1) 0.17 T(5)N(2)D(1) 100 E E(80)A(4)Q(8) 0.18 F(2)V(1)T(1).(1) 26 F Y(43)F(25)L(22) 0.19 H(1).(2)G(2) S(1) 107 W L(35)W(36)F(4) 0.20.(22)Q(1) 36 T L(61)T(18)M(4) 0.21 C(9)V(1)W(2) I(1) 84 E E(59)I(2)D(12) 0.22 S(2)Q(4)T(14) N(1)R(1).(1) 56 D Y(39)N(12)V(2) 0.23 I(2)D(30)T(5) E(5) 86 D D(66)N(2)Y(4) 0.24 F(15)L(2)E(7) R(1) 27 N R(57)N(29)H(5) 0.25.(2)S(1)F(1) K(1) Table 8. Residues forming surface patch in 1bccF. Fig. 7. A possible active surface on the chain 1bccF. Table 8. continued res type substitutions(%) cvg 49 R R(91)L(4)M(1) 0.12 I(2) 103 E E(90)D(2)Q(1) 0.13 A(2).(1)N(1) 75 L L(90)E(2)P(2) 0.14 Table R (TD)(SYEVCLAPIG)(FMW)(N) 50 L (YR)(H)(TKE)(SQCDG) 35 D (R)(H)(FYW)(KCG) 89 Y (K)(QR)(M)(E) 31 L (R)(YE)(K)(T) 30 G (KR)(E)(FMWH)(Q) 65 A (KR)(E)(QH)(Y) 58 R (D)(T)(E)(Y) 34 D (R)(FW)(VCAHG)(Y) 49 R (T)(Y)(D)(SCG) 103 E (H)(FW)(Y)(R) 75 L (R)(Y)(H)(T) 64 R (TY)(D)(FW)(VA) 92 P (R)(Y)(H)(T) 51 P (R)(Y)(H)(T) 100 E (H)(FW)(R)(Y) 6

7 Table 9. continued 26 F (K)(E)(Q)(D) 107 W (E)(T)(K)(D) 36 T (R)(K)(H)(Q) 84 E (FWH)(Y)(R)(VCAG) 56 D (R)(H)(FW)(K) 86 D (R)(H)(FW)(CG) 27 N (Y)(T)(FW)(E) Table 9. Disruptive for the surface patch in 1bccF. Fig. 8. Residues in 1bccA colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) 3 CHAIN 1BCCA 3.1 P31800 overview From SwissProt, id P31800, 90% identical to 1bccA: Description: Ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial precursor (EC ). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This protein may mediate formation of the complex between cytochromes c and c1. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane; matrix side. Similarity: Belongs to the peptidase M16 family. Caution: Does not seem to have a protease activity as it lack the zinc-binding site. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 3.2 Multiple sequence alignment for 1bccA For the chain 1bccA, the alignment 1bccA.msf (attached) with 150 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccA.msf. Its statistics, from the alistat program are the following: Fig. 9. Residues in 1bccA colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Format: MSF Number of sequences: 150 Total number of residues: Smallest: 367 Largest: 442 Average length: Alignment length: 442 Average identity: 38% Most related pair: 99% Most unrelated pair: 15% Most distant seq: 43% Furthermore, <1% of residues show as conserved in this alignment. The alignment consists of 39% eukaryotic ( 8% vertebrata, 2% arthropoda, 13% fungi, 4% plantae), and 2% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccA.descr. 3.3 Residue ranking in 1bccA The 1bccA sequence is shown in Figs. 8 9, with each residue colored according to its estimated importance. The full listing of residues in 1bccA can be found in the file called 1bccA.ranks sorted in the attachment. 7

8 3.4 Top ranking residues in 1bccA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein. Figure 10 shows residues in 1bccA colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 11. Residues in 1bccA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Fig. 10. Residues in 1bccA, colored by their relative importance. Clockwise: front, back, top and bottom views. Table 10. continued cluster size member color residues blue 6 353,356,357,358,362,399 yellow 2 270,272 Table 10. Clusters of top ranking residues in 1bccA Clustering of residues at 25% coverage. Fig. 11 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.11 are composed of the residues listed in Table 10. Table 10. cluster size member color residues red 90 19,22,24,26,28,37,38,40,44 45,46,48,53,54,56,57,58,59 60,61,62,63,64,65,66,67,70 75,76,80,83,84,85,86,87,88 90,92,93,95,97,98,99,105, ,115,116,130,131,133, ,138,149,150,151,153, ,169,170,190,194,195, ,199,200,201,216,236, ,259,310,314,316,318, ,369,370,371,377,378, ,381, Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccI.Table 11 lists the top 25% of residues at the interface with 1bccI. The following table (Table 12) suggests possible disruptive replacements for these residues (see Section Table E E(59) / A(27) L(1)K V(7).(3)I 80 E N(1) / D(5) E(88).(3)QG 64 F H(1) /

9 Table 11. continued F(92) L(1) Y(1)S A(2)VG 85 H K(1) / H(54) Y(15) Q(7) R(1) S(1) I(1) E(3) D(8) L(3) N(2) 65 K K(90) / S(2) L(3)R N(2)HT 86 L L(71) / I(17) F(4) C(1) V(3)MT 84 A A(41) / G(48) V(2) S(5)FI 87 N N(85) / E(2) D(2)V T(3) S(4) 280 Y W(38) / (22)V F(12) Y(23) G(1)X Table 11. The top 25% of residues in 1bccA at the interface with 1bccI. Table E (H)(Y)(FW)(R) 80 E (FWH)(Y)(R)(VA) 64 F (K)(E)(Q)(D) 85 H (E)(T)(MD)(CG) Table 12. continued 65 K (Y)(FW)(T)(VA) 86 L (R)(Y)(H)(K) 84 A (KR)(E)(Y)(Q) 87 N (Y)(H)(FW)(R) 280 Y (K)(Q)(E)(M) Table 12. List of disruptive for the top 25% of residues in 1bccA, that are at the interface with 1bccI. Fig. 12. Residues in 1bccA, at the interface with 1bccI, colored by their relative importance. 1bccI is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccA.) Figure 12 shows residues in 1bccA colored by their importance, at the interface with 1bccI. Interface with 1bccB.Table 13 lists the top 25% of residues at the interface with 1bccB. The following table (Table 14) suggests Table G G(100) / E N(1) / D(5) E(88).(3)QG 85 H K(1) /

10 Table 13. continued H(54) Y(15) Q(7) R(1) S(1) I(1) E(3) D(8) L(3) N(2) 362 R R(10) / K(83) L(2)Q M(3)H 370 D E(40) / D(52)C N(4) S(2)AH 356 R M(1) / R(84)E Q(4) I(2)S L(3) A(1)HG 371 G T(2) / S(41) G(48)K D(6) Table 13. The top 25% of residues in 1bccA at the interface with 1bccB. Table G (KER)(FQMWHD)(NYLPI)(SVA) 80 E (FWH)(Y)(R)(VA) 85 H (E)(T)(MD)(CG) 362 R (T)(Y)(D)(S) 370 D (R)(FWH)(KY)(VCAG) 356 R (TY)(D)(E)(CG) 371 G (R)(K)(FW)(H) Table 14. List of disruptive for the top 25% of residues in 1bccA, that are at the interface with 1bccB. Fig. 13. Residues in 1bccA, at the interface with 1bccB, colored by their relative importance. 1bccB is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccA.) Interface with 1bccE.Table 15 lists the top 25% of residues at the interface with 1bccE. The following table (Table 16) suggests Table G G(96) / C(1) A(2) 236 F F(80) / Y(13).(2) V(1)XLW H 151 N D(81) / E(14) N(1) S(2) 170 P T(22) / P(69) E(1)L S(4) A(1) 138 L L(6) / I(33) Q(12) M(32)N Figure 13 shows residues in 1bccA colored by their importance, at the interface with 1bccB. 10

11 Table 15. continued S(9) A(4)KV Table 15. The top 25% of residues in 1bccA at the interface with 1bccE. Table G (KER)(QHD)(FMW)(Y) 236 F (KE)(Q)(D)(T) 151 N (Y)(FWH)(R)(T) 170 P (R)(Y)(H)(K) 138 L (Y)(R)(H)(T) Table 16. List of disruptive for the top 25% of residues in 1bccA, that are at the interface with 1bccE. Interface with 1bccG.Table 17 lists the top 25% of residues at the interface with 1bccG. The following table (Table 18) suggests Table F F(80) / Y(13).(2) V(1)XLW H 238 G G(86) / A(3)SX R(2) K(2) P(2)V.C 239 S S(54) / G(35) Q(1) N(1)XA D(2) C(1)V R(1) Table 17. The top 25% of residues in 1bccA at the interface with 1bccG. Table F (KE)(Q)(D)(T) 238 G (E)(R)(K)(D) 239 S (R)(K)(H)(FW) Table 18. List of disruptive for the top 25% of residues in 1bccA, that are at the interface with 1bccG. Figure 15 shows residues in 1bccA colored by their importance, at the interface with 1bccG. Fig. 14. Residues in 1bccA, at the interface with 1bccE, colored by their relative importance. 1bccE is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccA.) Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bccA surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 16. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 19, while Table 20 suggests possible disruptive replacements for these residues (see Section Figure 14 shows residues in 1bccA colored by their importance, at the interface with 1bccE. 11

12 Fig. 15. Residues in 1bccA, at the interface with 1bccG, colored by their relative importance. 1bccG is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccA.) Fig. 16. A possible active surface on the chain 1bccA. The larger cluster it belongs to is shown in blue. Table 19. res type substitutions(%) cvg 44 G G(100) G G(100) E E(94)AD(4)G G G(96)C(1)A(2) L L(96).(2)V(1) G G(94).(2)K(3) E D(9)E(90) R R(86)Y(3)Q(3) 0.04 F(1).(4)H(1) 95 T T(90)N(4)SLQ(1) 0.04 M(2) 190 Y Y(89)L(6)HF(3) 0.04 Table 19. continued res type substitutions(%) cvg 57 Y H(81)PR(3)N(2) 0.05 Y(5)A(3)F(2) 131 R R(76)Q(2)K(19) 0.05 L(2) 194 R R(71)Q(11)H(2) 0.05 D(7)N(4)EGT 46 R R(90)T(1)VSP 0.06 I(2)K(3) 88 A S(5)A(90)V(2) 0.06 C(1)T 115 D D(93)S(2)NE(2)Q H R(4)H(86)K(5) 0.07 N(2)ES 90 S T(84)V(4)S(5) 0.07 N(2)A(2) 92 R R(87)IT(5)V(2)H 0.07 K(3) 150 F F(80)M(7)L(9) 0.07 G(1)HT 76 E E(59)A(27)L(1)K 0.08 V(7).(3)I 137 E E(93)D(2)Q(2)TA F F(76)Q(14)Y(5) 0.08 S(2)T(1)X 60 E E(93)PSA(2)T(1) 0.09 Q(1) 67 T T(94)L(1)VS(2)H Y Y(91)F(4)H(2)X 0.09 W(1)G 56 G A(71)S(18)RT(1) 0.10 G(7) 75 L L(53)I(38)V(2)T 0.10.(4)M(1) 97 Y V(2)Y(84)F(5) 0.10 L(3)I(4) 378 D F(1)Q(11)D(58) 0.10 R(17)GE(8)NL. 116 I V(12)I(77)L(5)T 0.11 S(2)CK 151 N D(81)E(14)N(1) 0.11 S(2) 53 N N(75)RA(5)S(8) 0.12 H(8)Y 80 E N(1)D(5)E(88) 0.12.(3)QG 105 D D(84)N(7)HA(1) 0.12 Q(1)Y(2)K(1)G 113 L L(78)I(14)V(7) F H(1)F(92)L(1) 0.13 Y(1)SA(2)VG 66 G G(90)TS(5)A(1)N 0.13 QHK 12

13 Table 19. continued res type substitutions(%) cvg 85 H K(1)H(54)Y(15) 0.14 Q(7)R(1)S(1) I(1)E(3)D(8) L(3)N(2) 130 E E(85)A(6)T(1) 0.14 Q(4)HSV 370 D E(40)D(52)CN(4) 0.14 S(2)AH 24 R R(57)T(32).(2) 0.15 V(1)K(6)S 65 K K(90)S(2)L(3)R 0.16 N(2)HT 98 Y F(4)Y(83)A(1) 0.16 L(2)NT(3)I(2)V 170 P T(22)P(69)E(1)L 0.17 S(4)A(1) 371 G T(2)S(41)G(48)K 0.17 D(6) 377 E G(2)E(84)K(2) 0.17 D(4)S(3)N(2)T. 45 S S(71)T(4)G(17)P 0.18 C(1)A(5) 154 H H(67)Q(18)N(9) 0.18 L(2)TY(1)A 316 E D(85)E(6)N(4)X 0.18 H(2)Q 28 E E(76)D(10)LQ(4) 0.19 RI(2)V(3).(1)G 86 L L(71)I(17)F(4) 0.19 C(1)V(3)MT 259 G G(69)A(10)I(2) 0.19 T(5)S(7)XV(2)C 37 V V(68)L(15)R 0.20 I(15)M 109 A V(5)A(70)M(5) 0.20 C(7)G(3)S(2) N(3)LT(1) 369 L L(78)R(2)M(4) 0.20 Y(1)AI(4)F(3) E(1)NT(1)V(1) 381 R K(4)R(91)N(1)EA 0.20.ST 138 L L(6)I(33)Q(12) 0.22 M(32)NS(9)A(4)K V 200 A V(11)A(66)S(16) 0.22 T(4) 84 A A(41)G(48)V(2) 0.23 S(5)FI 164 A A(10)G(81)P(2) 0.23 S(4)R 216 F F(82)L(14)SXI Table 19. continued res type substitutions(%) cvg 62 L L(28)M(62)I(2) 0.24 C(3)T(1)VS 87 N N(85)E(2)D(2)V 0.24 T(3)S(4) 195 M M(78)L(2)V(4) 0.24 I(10)T(3)AS 133 V N(2)V(81)T(4) 0.25 M(2)R(1)A(4)S K(3)Y 177 L I(42)M(9)F(24) 0.25 V(4)L(18). Table 19. Residues forming surface patch in 1bccA. Table G (KER)(FQMWHD)(NYLPI)(SVA) 83 G (KER)(FQMWHD)(NYLPI)(SVA) 48 E (H)(FWR)(Y)(KVCAG) 169 G (KER)(QHD)(FMW)(Y) 19 L (Y)(R)(H)(T) 22 G (E)(FW)(R)(D) 93 E (FWH)(R)(YVCAG)(T) 70 R (T)(D)(E)(VCAG) 95 T (R)(H)(FW)(K) 190 Y (K)(Q)(E)(M) 57 Y (K)(E)(Q)(M) 131 R (T)(Y)(D)(SCG) 194 R (T)(Y)(D)(VA) 46 R (Y)(D)(T)(E) 88 A (KR)(E)(Y)(QH) 115 D (R)(FWH)(Y)(VCAG) 61 H (TE)(M)(D)(VCAG) 90 S (R)(K)(H)(FQW) 92 R (D)(T)(E)(Y) 150 F (E)(K)(D)(Q) 76 E (H)(Y)(FW)(R) 137 E (H)(FW)(R)(Y) 310 F (K)(E)(Q)(DR) 60 E (H)(FW)(R)(Y) 67 T (K)(R)(Q)(EMH) 314 Y (K)(Q)(E)(M) 56 G (K)(E)(R)(QM) 75 L (R)(Y)(H)(T) 97 Y (K)(QR)(E)(M) 378 D (R)(H)(FW)(Y) 116 I (R)(Y)(H)(KE) 151 N (Y)(FWH)(R)(T) 53 N (Y)(E)(T)(FW) 13

14 Table 20. continued 80 E (FWH)(Y)(R)(VA) 105 D (R)(FW)(H)(Y) 113 L (YR)(H)(T)(KE) 64 F (K)(E)(Q)(D) 66 G (E)(R)(K)(FW) 85 H (E)(T)(MD)(CG) 130 E (H)(FW)(R)(Y) 370 D (R)(FWH)(KY)(VCAG) 24 R (D)(Y)(T)(E) 65 K (Y)(FW)(T)(VA) 98 Y (K)(R)(Q)(E) 170 P (R)(Y)(H)(K) 371 G (R)(K)(FW)(H) 377 E (FW)(H)(R)(Y) 45 S (R)(K)(H)(Q) 154 H (E)(D)(M)(TQ) 316 E (FW)(H)(Y)(VCAG) 28 E (H)(FW)(Y)(R) 86 L (R)(Y)(H)(K) 259 G (R)(K)(E)(H) 37 V (Y)(ER)(K)(H) 109 A (R)(KY)(E)(H) 369 L (R)(Y)(H)(T) 381 R (Y)(TD)(FW)(EVCLAPIG) 138 L (Y)(R)(H)(T) 200 A (KR)(E)(Y)(QH) 84 A (KR)(E)(Y)(Q) 164 A (E)(Y)(KR)(D) 216 F (K)(E)(QR)(T) 62 L (R)(Y)(H)(K) 87 N (Y)(H)(FW)(R) 195 M (Y)(H)(R)(T) 133 V (Y)(E)(R)(K) 177 L (YR)(T)(H)(KE) Table 20. Disruptive for the surface patch in 1bccA. Another group of surface residues is shown in Fig.17. The residues belonging to this surface patch are listed in Table 21, while Table 22 suggests possible disruptive replacements for these residues (see Section Table 21. res type substitutions(%) cvg 358 K K(72)R(25)SC(1) E E(83)A(1)Q(3) 0.11 D(8)TL(1)RI 362 R R(10)K(83)L(2)Q 0.14 M(3)H 356 R M(1)R(84)EQ(4) 0.16 I(2)SL(3)A(1)HG 399 L I(71)V(16)L(10) 0.21.(1) Fig. 17. Another possible active surface on the chain 1bccA. Table 21. continued res type substitutions(%) cvg 361 L L(64)M(23)Y(1) 0.25 I(1)V(3)T(2) F(2)A Table 21. Residues forming surface patch in 1bccA. Table K (Y)(FW)(T)(VAD) 353 E (H)(FW)(Y)(R) 362 R (T)(Y)(D)(S) 356 R (TY)(D)(E)(CG) 399 L (YR)(H)(T)(KE) 361 L (R)(Y)(KH)(E) Table 22. Disruptive for the surface patch in 1bccA. 4 CHAIN 1BCCJ 4.1 P00130 overview From SwissProt, id P00130, 98% identical to 1bccJ: Description: Ubiquinol-cytochrome c reductase complex 7.2 kda protein (EC ) (Cytochrome C1, nonheme 7 kda protein) (Complex III subunit X). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. 14

15 Fig. 18. Residues 4-62 in 1bccJ colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) 4.4 Top ranking residues in 1bccJ and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein. Figure 19 shows residues in 1bccJ colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This subunit interacts with cytochrome c1. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane. Similarity: Belongs to the UQCR10/QCR9 family. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4.2 Multiple sequence alignment for 1bccJ For the chain 1bccJ, the alignment 1bccJ.msf (attached) with 30 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccJ.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 30 Total number of residues: 1653 Smallest: 49 Largest: 59 Average length: 55.1 Alignment length: 59 Average identity: 47% Most related pair: 98% Most unrelated pair: 24% Most distant seq: 60% Furthermore, 3% of residues show as conserved in this alignment. The alignment consists of 53% eukaryotic ( 13% vertebrata, 10% arthropoda, 23% fungi, 3% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccJ.descr. 4.3 Residue ranking in 1bccJ The 1bccJ sequence is shown in Fig. 18, with each residue colored according to its estimated importance. The full listing of residues in 1bccJ can be found in the file called 1bccJ.ranks sorted in the attachment. Fig. 19. Residues in 1bccJ, colored by their relative importance. Clockwise: front, back, top and bottom views Clustering of residues at 25% coverage. Fig. 20 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.20 are composed of the residues listed in Table 23. Table 23. cluster size member color residues red 9 47,49,50,51,52,53,54,55,56 blue 3 31,32,36 yellow 2 16,17 Table 23. Clusters of top ranking residues in 1bccJ Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccD.Table 24 lists the top 25% of residues at the interface with 1bccD. The following table (Table 25) suggests 15

16 Table N (Y)(FTWH)(SEVCARG)(MD) 49 G (KER)(FQMWHD)(NYLPI)(SVA) 31 F (KE)(QD)(R)(TN) 52 W (K)(E)(Q)(D) 36 D (R)(FWH)(YVCAG)(K) 50 K (Y)(T)(FW)(SVCAG) 55 I (YR)(H)(TKE)(SQCDG) 51 L (Y)(T)(H)(R) Table 25. List of disruptive for the top 25% of residues in 1bccJ, that are at the interface with 1bccD. Fig. 20. Residues in 1bccJ, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table N N(100) / G G(100) / F F(93) / G(6) 52 W W(93) / Y(6) 36 D D(96) / E(3) 50 K K(76) / R(23) 55 I I(90) / V(10) 51 L L(56) / Q(36) R(6) Table 24. The top 25% of residues in 1bccJ at the interface with 1bccD. Fig. 21. Residues in 1bccJ, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccJ.) Figure 21 shows residues in 1bccJ colored by their importance, at the interface with 1bccD. PEE binding site. Table 26 lists the top 25% of residues at the interface with 1bccPEE6 (pee). The following table (Table 27) suggests Table E E(80) / D(3) Q(13) 16

17 Table 26. continued S(3) 17 T T(50) / A(3) N(43) S(3) Table 26. The top 25% of residues in 1bccJ at the interface with PEE.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table E (FWH)(YR)(VCAG)(T) 17 T (R)(K)(H)(FW) Table 27. List of disruptive for the top 25% of residues in 1bccJ, that are at the interface with PEE. Interface with 1bccE.Table 28 lists the top 25% of residues at the interface with 1bccE. The following table (Table 29) suggests Table F F(93) / G(6) 36 D D(96) / E(3) 32 E E(80) / D(3) Q(13) S(3) 10 Y Y(83) / N(6).(6) E(3) Table 28. The top 25% of residues in 1bccJ at the interface with 1bccE. Table F (KE)(QD)(R)(TN) 36 D (R)(FWH)(YVCAG)(K) 32 E (FWH)(YR)(VCAG)(T) 10 Y (K)(VMA)(QR)(LPI) Table 29. List of disruptive for the top 25% of residues in 1bccJ, that are at the interface with 1bccE. Figure 23 shows residues in 1bccJ colored by their importance, at the interface with 1bccE. Interface with 1bccA.Table 30 lists the top 25% of residues at the interface with 1bccA. The following table (Table 31) suggests Fig. 22. Residues in 1bccJ, at the interface with PEE, colored by their relative importance. The ligand (PEE) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccJ.) Figure 22 shows residues in 1bccJ colored by their importance, at the interface with 1bccPEE6. Table R R(96) / N(3) 17 T T(50) / A(3) N(43) S(3) 10 Y Y(83) /

18 Fig. 23. Residues in 1bccJ, at the interface with 1bccE, colored by their relative importance. 1bccE is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccJ.) Fig. 24. Residues in 1bccJ, at the interface with 1bccA, colored by their relative importance. 1bccA is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccJ.) Table 30. continued N(6).(6) E(3) Table 30. The top 25% of residues in 1bccJ at the interface with 1bccA. Table R (T)(YD)(SEVCAG)(FLWPI) 17 T (R)(K)(H)(FW) 10 Y (K)(VMA)(QR)(LPI) Table 31. List of disruptive for the top 25% of residues in 1bccJ, that are at the interface with 1bccA. Figure 24 shows residues in 1bccJ colored by their importance, at the interface with 1bccA. Fig. 25. A possible active surface on the chain 1bccJ. belonging to this surface patch are listed in Table 32, while Table 33 suggests possible disruptive replacements for these residues (see Section Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bccJ surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 25. The residues 18

19 Table 32. res type substitutions(%) cvg 47 N N(100) G G(100) W W(93)Y(6) K K(76)R(23) K K(83)D(10)E(6) H D(76)H(23) I I(90)V(10) L L(56)Q(36)R(6) K K(73)R(16).(6) 0.22 E(3) Table 32. Residues forming surface patch in 1bccJ. Table N (Y)(FTWH)(SEVCARG)(MD) 49 G (KER)(FQMWHD)(NYLPI)(SVA) 52 W (K)(E)(Q)(D) 50 K (Y)(T)(FW)(SVCAG) 53 K (Y)(FW)(T)(VCAG) 54 H (TEQM)(KVCAG)(SNLPDI)(R) 55 I (YR)(H)(TKE)(SQCDG) 51 L (Y)(T)(H)(R) 56 K (Y)(FTW)(VCAG)(S) Table 33. Disruptive for the surface patch in 1bccJ. 5 CHAIN 1BCCE 5.1 P13272 overview From SwissProt, id P13272, 93% identical to 1bccE: Description: Ubiquinol-cytochrome c reductase iron-sulfur subunit, mitochondrial precursor (EC ) (Rieske iron-sulfur protein) (RISP) [Contains: Ubiquinol-cytochrome c reductase 8 kda protein (Complex III subunit IX)]. Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: Component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is a respiratory chain that generates an electrochemical potential coupled to ATP synthesis. Function: The transit peptide of the Rieske protein seems to form part of the bc1 complex in beef and is considered to be the subunit IX of that complex. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Cofactor: Binds 1 2Fe-2S iron sulfur cluster per subunit. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane. Fig. 26. Residues in 1bccE colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Miscellaneous: The Rieske protein is a high potential 2Fe-2S protein. Similarity: Contains 1 Rieske domain. Caution: Ref.1 sequence differs from that shown by extensive frameshifts and probable sequencing errors in the region of the transit peptide (1-78). About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 5.2 Multiple sequence alignment for 1bccE For the chain 1bccE, the alignment 1bccE.msf (attached) with 240 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccE.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 240 Total number of residues: Smallest: 148 Largest: 196 Average length: Alignment length: 196 Average identity: 46% Most related pair: 99% Most unrelated pair: 15% Most distant seq: 51% Furthermore, <1% of residues show as conserved in this alignment. The alignment consists of 26% eukaryotic ( 10% vertebrata, 1% arthropoda, 5% fungi, 4% plantae), and 12% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccE.descr. 5.3 Residue ranking in 1bccE The 1bccE sequence is shown in Fig. 26, with each residue colored according to its estimated importance. The full listing of residues 19

20 in 1bccE can be found in the file called 1bccE.ranks sorted in the attachment. 5.4 Top ranking residues in 1bccE and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein. Figure 27 shows residues in 1bccE colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 28. Residues in 1bccE, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccC.Table 35 lists the top 25% of residues at the interface with 1bccC. The following table (Table 36) suggests Fig. 27. Residues in 1bccE, colored by their relative importance. Clockwise: front, back, top and bottom views Clustering of residues at 25% coverage. Fig. 28 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.28 are composed of the residues listed in Table 34. Table 34. cluster size member color residues red 40 91,92,93,94,95,97,117, ,137,139,140,141,142, ,146,151,155,156,157, ,160,161,162,163,165, ,169,170,172,174,175, ,179,180,183 blue 3 84,101,102 Table 34. Clusters of top ranking residues in 1bccE. Table A A(35) / P(46)L D(2)Q S(9) I(2)TYH M Table 35. The top 25% of residues in 1bccE at the interface with 1bccC. Table A (R)(Y)(K)(E) 20

21 Table 36. continued type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table 36. List of disruptive for the top 25% of residues in 1bccE, that are at the interface with 1bccC. Table C (KER)(FQMWHD)(NYLPI)(SVA) 139 C (KR)(E)(FQMWH)(D) 141 H (E)(T)(QD)(SKMCG) 143 G (KR)(E)(FQMWH)(D) 158 C (KER)(FQMWHD)(NLPI)(Y) 160 C (KER)(FQMWHD)(NLPI)(Y) 161 H (E)(TQMD)(SNVCLAPIG)(K) 162 G (KER)(FQMWHD)(NLPI)(Y) 163 S (KR)(FQMWH)(NLPI)(YE) 142 L (R)(Y)(T)(KE) Table 38. List of disruptive for the top 25% of residues in 1bccE, that are at the interface with FES. Fig. 29. Residues in 1bccE, at the interface with 1bccC, colored by their relative importance. 1bccC is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccE.) Figure 29 shows residues in 1bccE colored by their importance, at the interface with 1bccC. FES binding site. Table 37 lists the top 25% of residues at the interface with 1bccFES4 (fes). The following table (Table 38) suggests Table 37. antn 144 C C(100) / S-S 139 C C(99)S / site 141 H H(99)L / site 143 G G(99)T / C C(99) / site 160 C C(99) / S-S 161 H H(99) / G G(99) / S S(99) / L L(99)FA / Table 37. The top 25% of residues in 1bccE at the interface with FES.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each Fig. 30. Residues in 1bccE, at the interface with FES, colored by their relative importance. The ligand (FES) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccE.) Figure 30 shows residues in 1bccE colored by their importance, at the interface with 1bccFES4. Interface with 1bccD.Table 39 lists the top 25% of residues at the interface with 1bccD. The following table (Table 40) suggests 21

22 Table A A(95) / G(2)PSE Table 39. The top 25% of residues in 1bccE at the interface with 1bccD. Table A (R)(K)(Y)(H) Table 40. List of disruptive for the top 25% of residues in 1bccE, that are at the interface with 1bccD. Fig. 31. Residues in 1bccE, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccE.) Figure 31 shows residues in 1bccE colored by their importance, at the interface with 1bccD Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bccE surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 32. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 41, 22

23 Fig. 32. A possible active surface on the chain 1bccE. The larger cluster it belongs to is shown in blue. Table 41. continued res type substitutions(%) cvg antn HIA 172 R R(79)Y(6)I(1) 0.23 F(7)Q(1)LV(1). 94 K K(83)GAQ(11) 0.24 R(2)H(1) 151 G G(86)E(1)K(2) 0.24 D(8)SRL 73 K S(32)P(24)K(27) 0.25 Q(1)T(11)N(2)IE A Table 41. Residues forming surface patch in 1bccE. while Table 42 suggests possible disruptive replacements for these residues (see Section Table 41. res type substitutions(%) cvg antn 144 C C(100) 0.01 S-S 139 C C(99)S 0.06 site 140 T T(99)S H H(99)L 0.06 site 143 G G(99)T P P(99)Q C C(99) S-S 161 H H(99) G G(99) L L(99)FA P P(99)L D D(99) R R(98)G P P(99) N N(98).(1) A A(95)G(2)PSE P P(95)V(2).(1) P P(95)HR.Q(2)S P P(97)SL.TH G G(94).(5) W W(90)F(7)YI G G(93)N(1)K(2) 0.16 E(1)DS 174 G G(93)V(2)Q(3) R R(75)Q(14)L(8)N 0.18 G 126 R R(79)N(13)AH 0.18 Q(1)G(1)TLEMDV 97 F F(83)W(11)Y(3)V 0.19 L 119 D D(94)H(3)REMP L L(88)M(5)TQV(1) 0.21 F(1)RAK 157 Y F(75)Y(21)L(1)S 0.21 Table C (KER)(FQMWHD)(NYLPI)(SVA) 139 C (KR)(E)(FQMWH)(D) 140 T (KR)(FQMWH)(NELPI)(D) 141 H (E)(T)(QD)(SKMCG) 143 G (KR)(E)(FQMWH)(D) 146 P (Y)(THR)(SCG)(FEW) 160 C (KER)(FQMWHD)(NLPI)(Y) 161 H (E)(TQMD)(SNVCLAPIG)(K) 162 G (KER)(FQMWHD)(NLPI)(Y) 142 L (R)(Y)(T)(KE) 95 P (YR)(TH)(SKECG)(FQWD) 166 D (R)(FWH)(VCAG)(KY) 170 R (D)(ELPI)(T)(VA) 175 P (YR)(TH)(SCG)(KE) 179 N (Y)(FTWH)(SVCAG)(ER) 70 A (R)(K)(Y)(H) 183 P (Y)(R)(H)(T) 159 P (Y)(T)(R)(H) 177 P (R)(Y)(H)(K) 196 G (KER)(FQMWHD)(NLPI)(Y) 91 W (K)(E)(Q)(TD) 93 G (R)(FW)(H)(K) 174 G (ER)(KH)(FWD)(Y) 92 R (T)(Y)(D)(E) 126 R (Y)(T)(D)(E) 97 F (K)(E)(Q)(D) 119 D (R)(FWH)(Y)(CG) 117 L (Y)(R)(T)(H) 157 Y (K)(Q)(ER)(M) 172 R (T)(D)(E)(Y) 94 K (Y)(T)(FW)(SD) 151 G (R)(FW)(H)(K) 73 K (Y)(FW)(T)(H) Table 42. Disruptive for the surface patch in 1bccE. 23

24 6 CHAIN 1BCCB 6.1 P23004 overview From SwissProt, id P23004, 84% identical to 1bccB: Description: Ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial precursor (EC ) (Complex III subunit II). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. The core protein 2 is required for the assembly of the complex. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane; matrix side. Similarity: Belongs to the peptidase M16 family. Caution: Does not seem to have a protease activity as it lack the zinc-binding site. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 6.2 Multiple sequence alignment for 1bccB For the chain 1bccB, the alignment 1bccB.msf (attached) with 14 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccB.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 14 Total number of residues: 5610 Smallest: 387 Largest: 406 Average length: Alignment length: 406 Average identity: 58% Most related pair: 99% Most unrelated pair: 21% Most distant seq: 30% Furthermore, 9% of residues show as conserved in this alignment. The alignment consists of 57% eukaryotic ( 50% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccB.descr. 6.3 Residue ranking in 1bccB The 1bccB sequence is shown in Figs , with each residue colored according to its estimated importance. The full listing of residues Fig. 33. Residues in 1bccB colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Fig. 34. Residues in 1bccB colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) in 1bccB can be found in the file called 1bccB.ranks sorted in the attachment. 6.4 Top ranking residues in 1bccB and their position on the structure In the following we consider residues ranking among top 23% of residues in the protein (the closest this analysis allows us to get to 25%). Figure 35 shows residues in 1bccB colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment Clustering of residues at 23% coverage. Fig. 36 shows the top 23% of all residues, this time colored according to clusters they belong to. The clusters in Fig.36 are composed of the residues listed in Table 43. Table 43. cluster size member color residues red 57 36,48,53,54,55,58,64,68,69 102,103,107,121,123,164, ,198,200,203,205,206, ,241,242,258,260,262,263 24

25 Table 43. continued cluster size member color residues 384,385,387,390,422,423 blue 6 27,28,29,31,32,213 yellow 5 86,87,91,93,94 green 3 135,136,139 purple 2 268,416 azure 2 145,147 turquoise 2 221,223 brown 2 155,159 Table 43. Clusters of top ranking residues in 1bccB Overlap with known functional surfaces at 23% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccI.Table 44 lists the top 23% of residues at the interface with 1bccI. The following table (Table 45) suggests possible disruptive replacements for these residues (see Section Fig. 35. Residues in 1bccB, colored by their relative importance. Clockwise: front, back, top and bottom views. Table D E(85) / D(14) Table 44. The top 23% of residues in 1bccB at the interface with 1bccI. Table D (R)(FWH)(YVCAG)(K) Table 45. List of disruptive for the top 23% of residues in 1bccB, that are at the interface with 1bccI. Fig. 36. Residues in 1bccB, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 43. continued cluster size member color residues 274,275,276,311,312,320, ,352,357,359,360,361, ,373,374,378,380,381,383 Figure 37 shows residues in 1bccB colored by their importance, at the interface with 1bccI. Interface with 1bccA.Table 46 lists the top 23% of residues at the interface with 1bccA. The following table (Table 47) suggests Table G G(100) / G G(100) / L L(100) /

26 Table G (KER)(FQMWHD)(NYLPI)(SVA) 94 G (KER)(FQMWHD)(NYLPI)(SVA) 369 L (YR)(TH)(SKECG)(FQWD) 87 R (TD)(E)(SCG)(YVLAPI) 91 A (KR)(E)(YQH)(D) 373 E (H)(FYWR)(CG)(TKVA) 374 S (K)(QMR)(E)(FNLWPI) 86 T (KR)(QH)(FEMW)(N) 113 R (T)(YD)(SVCAG)(FELWPI) 147 D (R)(FWH)(YVCAG)(K) 359 A (YE)(D)(K)(QHR) Table 47. List of disruptive for the top 23% of residues in 1bccB, that are at the interface with 1bccA. Fig. 37. Residues in 1bccB, at the interface with 1bccI, colored by their relative importance. 1bccI is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccB.) Table 46. continued 87 R R(92) / W(7) 91 A A(92) / T(7) 373 E E(92) / A(7) 374 S S(92) / H(7) 86 T V(92) / T(7) 113 R K(85) / R(14) 147 D E(85) / D(14) 359 A R(85) / A(14) Table 46. The top 23% of residues in 1bccB at the interface with 1bccA. Fig. 38. Residues in 1bccB, at the interface with 1bccA, colored by their relative importance. 1bccA is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccB.) Figure 38 shows residues in 1bccB colored by their importance, at the interface with 1bccA Possible novel functional surfaces at 23% coverage. One group of residues is conserved on the 1bccB surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 39. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 48, while Table 49 suggests possible disruptive replacements for these residues (see Section 26

27 Fig. 39. A possible active surface on the chain 1bccB. The larger cluster it belongs to is shown in blue. Fig. 40. Another possible active surface on the chain 1bccB. The larger cluster it belongs to is shown in blue. Table 48. res type substitutions(%) cvg 27 T T(100) L L(100) N N(100) G G(100) A A(100) H H(100) K T(85)K(14) 0.23 Table 48. Residues forming surface patch in 1bccB. Table T (KR)(FQMWH)(NELPI)(D) 29 L (YR)(TH)(SKECG)(FQWD) 31 N (Y)(FTWH)(SEVCARG)(MD) 32 G (KER)(FQMWHD)(NYLPI)(SVA) 36 A (KYER)(QHD)(N)(FTMW) 213 H (E)(TQMD)(SNKVCLAPIG)(YR) 28 K (FYW)(TVAH)(SCDG)(ELPI) Table 49. Disruptive for the surface patch in 1bccB. Another group of surface residues is shown in Fig.40. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 50, while Table 51 suggests possible disruptive replacements for these residues (see Section Table 50. res type substitutions(%) cvg 58 E E(100) G G(100) L L(100) L L(100) Y Y(92)V(7) E E(92)H(7) 0.17 Table 50. Residues forming surface patch in 1bccB. 27

28 Table E (FWH)(YVCARG)(T)(SNKLPI) 64 G (KER)(FQMWHD)(NYLPI)(SVA) 69 L (YR)(TH)(SKECG)(FQWD) 123 L (YR)(TH)(SKECG)(FQWD) 107 Y (K)(Q)(EMR)(N) 121 E (FVCAWG)(TYHR)(SNKLPI)(QM) Table 51. Disruptive for the surface patch in 1bccB. Table 53. continued 260 E (FWH)(YVCARG)(T)(SNKLPI) 416 K (Y)(FTW)(SVCAG)(HD) 268 E (FW)(YH)(VCAG)(T) 422 K (Y)(T)(FW)(SCG) 262 A (YR)(KE)(H)(QD) 263 A (KER)(Y)(QHD)(N) Table 53. Disruptive for the surface patch in 1bccB. Another group of surface residues is shown in Fig.41. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Another group of surface residues is shown in Fig.42. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Fig. 41. Another possible active surface on the chain 1bccB. The larger cluster it belongs to is shown in blue. Fig. 42. Another possible active surface on the chain 1bccB. The larger cluster it belongs to is shown in blue. The residues belonging to this surface patch are listed in Table 52, while Table 53 suggests possible disruptive replacements for these residues (see Section Table 52. res type substitutions(%) cvg 239 Y Y(100) G G(100) G G(100) E E(100) K K(100) E E(92)K(7) K L(92)K(7) A P(85)A(14) A G(85)A(14) 0.23 Table 52. Residues forming surface patch in 1bccB. Table Y (K)(QM)(NEVLAPIR)(D) 241 G (KER)(FQMWHD)(NYLPI)(SVA) 242 G (KER)(FQMWHD)(NYLPI)(SVA) The residues belonging to this surface patch are listed in Table 54, while Table 55 suggests possible disruptive replacements for these residues (see Section Table 54. res type substitutions(%) cvg 102 R R(100) H H(100) V V(100) G G(100) A A(100) K K(100) L L(100) Q Q(100) E E(92)D(7) R R(92)N(7) Q Q(92)L(7) F F(92)Y(7) V V(92)I(7) K K(92)Y(7) E E(92)A(7) S S(92)H(7) E E(92)I(7) L L(92)F(7)

29 Table 54. continued res type substitutions(%) cvg 390 G G(92).(7) V L(92)V(7) H N(85)H(14) A R(85)A(14) F A(85)F(14) E D(85)E(14) S R(85)S(14) 0.23 Table 54. Residues forming surface patch in 1bccB. Table R (TD)(SYEVCLAPIG)(FMW)(N) 164 H (E)(TQMD)(SNKVCLAPIG)(YR) 274 V (KYER)(QHD)(N)(FTMW) 280 G (KER)(FQMWHD)(NYLPI)(SVA) 311 A (KYER)(QHD)(N)(FTMW) 361 K (Y)(FTW)(SVCAG)(HD) 369 L (YR)(TH)(SKECG)(FQWD) 385 Q (Y)(FTWH)(SVCAG)(D) 103 E (FWH)(R)(YVCAG)(T) 203 R (T)(YD)(SEVCAG)(FLWPI) 276 Q (Y)(TH)(FW)(SCG) 312 F (K)(E)(Q)(D) 352 V (YR)(KE)(H)(QD) 365 K (FTYVAW)(SCG)(D)(ELPHI) 373 E (H)(FYWR)(CG)(TKVA) 374 S (K)(QMR)(E)(FNLWPI) 380 E (H)(FYWR)(CG)(TVA) 387 L (R)(TY)(KE)(SCHG) 390 G (KER)(FQMWHD)(NLPI)(Y) 357 V (YR)(KE)(H)(QD) 198 H (E)(T)(MD)(SVQCAG) 359 A (YE)(D)(K)(QHR) 378 F (KE)(QD)(TR)(N) 381 E (FWH)(R)(YVCAG)(T) 384 S (FKMW)(YEQLPHIR)(ND)(VCAG) Table 55. Disruptive for the surface patch in 1bccB. Fig. 43. Residues in 1bccH colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) part of the mitochondrial respiratory chain. This protein may mediate formation of the complex between cytochromes c and c1. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane. Similarity: Belongs to the UQCRH/QCR6 family. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 7.2 Multiple sequence alignment for 1bccH For the chain 1bccH, the alignment 1bccH.msf (attached) with 45 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccH.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 45 Total number of residues: 2909 Smallest: 60 Largest: 66 Average length: 64.6 Alignment length: 66 Average identity: 42% Most related pair: 98% Most unrelated pair: 10% Most distant seq: 37% 7 CHAIN 1BCCH 7.1 P00126 overview From SwissProt, id P00126, 98% identical to 1bccH: Description: Ubiquinol-cytochrome c reductase complex 11 kda protein (EC ) (Mitochondrial hinge protein) (Cytochrome C1, nonheme 11 kda protein) (Complex III subunit VIII). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is Furthermore, <1% of residues show as conserved in this alignment. The alignment consists of 53% eukaryotic ( 13% vertebrata, 4% arthropoda, 17% fungi, 4% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccH.descr. 7.3 Residue ranking in 1bccH The 1bccH sequence is shown in Fig. 43, with each residue colored according to its estimated importance. The full listing of residues in 1bccH can be found in the file called 1bccH.ranks sorted in the attachment. 29

30 7.4 Top ranking residues in 1bccH and their position on the structure In the following we consider residues ranking among top 24% of residues in the protein (the closest this analysis allows us to get to 25%). Figure 44 shows residues in 1bccH colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 45. Residues in 1bccH, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Fig. 44. Residues in 1bccH, colored by their relative importance. Clockwise: front, back, top and bottom views Clustering of residues at 24% coverage. Fig. 45 shows the top 24% of all residues, this time colored according to clusters they belong to. The clusters in Fig.45 are composed of the residues listed in Table 56. Table 56. cluster size member color residues red 9 40,43,44,52,54,56,57,58,60 blue 4 24,66,68,69 yellow 2 77,78 Table 56. Clusters of top ranking residues in 1bccH Overlap with known functional surfaces at 24% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1bccD.Table 57 lists the top 24% of residues at the interface with 1bccD. The following table (Table 58) suggests Table 57. antn 54 C C(97) / S-S M(2) 40 C C(95) / S-S V(2).(2) 15 D D(97) / (2) 52 E E(84) / K(11) G(4) 56 E E(80) / Q(4) G(13) Y(2) 77 L L(93) / V(4) I(2) 78 K K(86) / (8) R(2) Q(2) 58 L L(37) / F(42) 30

31 Table 57. continued antn M(6) Y(13) 44 V V(88) / Q(4) I(4) L(2) 66 D D(68) / T(24) Q(2) N(4) 69 V V(55) / A(40) T(4) Table 57. The top 24% of residues in 1bccH at the interface with 1bccD. Table C (R)(KEH)(FYWD)(Q) 40 C (KER)(HD)(Q)(FMW) 15 D (R)(FWH)(VCAG)(KY) 52 E (FW)(H)(Y)(VAR) 56 E (FWH)(R)(VA)(Y) 77 L (YR)(H)(T)(KE) 78 K (Y)(T)(FW)(SVCAG) 58 L (R)(TY)(K)(EH) 44 V (Y)(R)(EH)(K) 66 D (R)(FWH)(Y)(VA) 69 V (KR)(E)(Y)(QH) Table 58. List of disruptive for the top 24% of residues in 1bccH, that are at the interface with 1bccD. Figure 46 shows residues in 1bccH colored by their importance, at the interface with 1bccD. Interface with 1bccG.Table 59 lists the top 24% of residues at the interface with 1bccG. The following table (Table 60) suggests Table E E(84) / K(11) G(4) 60 D D(53) / Fig. 46. Residues in 1bccH, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccH.) Table 59. continued H(42) E(4) 56 E E(80) / Q(4) G(13) Y(2) Table 59. The top 24% of residues in 1bccH at the interface with 1bccG. Table E (FW)(H)(Y)(VAR) 60 D (R)(FW)(VCAHG)(K) 56 E (FWH)(R)(VA)(Y) Table 60. List of disruptive for the top 24% of residues in 1bccH, that are at the interface with 1bccG. Figure 47 shows residues in 1bccH colored by their importance, at the interface with 1bccG. 31

32 62 suggests possible disruptive replacements for these residues (see Section Table 61. res type substitutions(%) cvg antn 54 C C(97)M(2) 0.01 S-S 40 C C(95)V(2).(2) 0.03 S-S 43 R R(95)K(2).(2) E E(84)Q(13)V(2) E E(84)K(11)G(4) D D(53)H(42)E(4) E E(80)Q(4)G(13) 0.15 Y(2) 58 L L(37)F(42)M(6) 0.20 Y(13) 44 V V(88)Q(4)I(4) 0.21 L(2) Table 61. Residues forming surface patch in 1bccH. Fig. 47. Residues in 1bccH, at the interface with 1bccG, colored by their relative importance. 1bccG is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccH.) Possible novel functional surfaces at 24% coverage. One group of residues is conserved on the 1bccH surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 48. The residues Table C (R)(KEH)(FYWD)(Q) 40 C (KER)(HD)(Q)(FMW) 43 R (T)(D)(Y)(VCAG) 57 E (H)(FW)(Y)(R) 52 E (FW)(H)(Y)(VAR) 60 D (R)(FW)(VCAHG)(K) 56 E (FWH)(R)(VA)(Y) 58 L (R)(TY)(K)(EH) 44 V (Y)(R)(EH)(K) Table 62. Disruptive for the surface patch in 1bccH. Fig. 48. A possible active surface on the chain 1bccH. belonging to this surface patch are listed in Table 61, while Table 8 CHAIN 1BCCC 8.1 Q7GTU5 overview From SwissProt, id Q7GTU5, 95% identical to 1bccC: Description: Cytochrome b. Organism, scientific name: Gallus gallus (Chicken). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Archosauria; Aves; Neognathae; Galliformes; Phasianidae; Phasianinae; Gallus. Function: Component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is a respiratory chain that generates an electrochemical potential coupled to ATP synthesis (By similarity). Cofactor: Binds two heme groups non-covalently (By similarity). Cofactor: Binds two heme groups non-covalently. Heme 1 (or BL or b562) is low-potential and absorbs at about 562 nm, and heme 2 (or BH or b566) is high-potential and absorbs at about 566 nm (By similarity). Subunit: The main subunits of complex b-c1 are: cytochrome b, cytochrome c1 and the Rieske protein (By similarity). Similarity: Belongs to the cytochrome b family. 32

33 Fig. 49. Residues in 1bccC colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) in 1bccC can be found in the file called 1bccC.ranks sorted in the attachment. 8.4 Top ranking residues in 1bccC and their position on the structure In the following we consider residues ranking among top 28% of residues in the protein (the closest this analysis allows us to get to 25%). Figure 51 shows residues in 1bccC colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 50. Residues in 1bccC colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) 8.2 Multiple sequence alignment for 1bccC For the chain 1bccC, the alignment 1bccC.msf (attached) with 181 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccC.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 181 Total number of residues: Smallest: 359 Largest: 379 Average length: Alignment length: 379 Average identity: 86% Most related pair: 99% Most unrelated pair: 40% Most distant seq: 48% Furthermore, 19% of residues show as conserved in this alignment. The alignment consists of 66% eukaryotic ( 66% vertebrata), and 1% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccC.descr. 8.3 Residue ranking in 1bccC The 1bccC sequence is shown in Figs , with each residue colored according to its estimated importance. The full listing of residues Fig. 51. Residues in 1bccC, colored by their relative importance. Clockwise: front, back, top and bottom views Clustering of residues at 28% coverage. Fig. 52 shows the top 28% of all residues, this time colored according to clusters they belong to. The clusters in Fig.52 are composed of the residues listed in Table 63. Table 63. cluster size member color residues red 64 46,48,49,52,53,56,84,87,88 91,92,122,126,127,129, ,134,139,140,141,143, ,148,149,150,152,153, ,162,166,167,168,170, ,175,176,178,180,183, ,187,256,265,266,269, ,273,274,276,280,281, ,291,292,336,340,359 33

34 Table 64. continued 209 P P(100) / W W(100) / Y Y(100) / Table 64. The top 28% of residues in 1bccC at the interface with PEE.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Fig. 52. Residues in 1bccC, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 63. continued cluster size member color residues blue 20 31,32,34,35,98,101,102, ,107,108,114,117,197, ,211,306,308,314 yellow 3 248,249,252 green 3 74,77,78 purple 2 319,320 azure 2 64,259 turquoise 2 326,327 Table W (KE)(TQD)(SNCRG)(M) 101 R (TD)(SYEVCLAPIG)(FMW)(N) 105 Y (K)(QM)(NEVLAPIR)(D) 209 P (YR)(TH)(SKECG)(FQWD) 273 W (KE)(TQD)(SNCRG)(M) 359 Y (K)(QM)(NEVLAPIR)(D) Table 65. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with PEE. Table 63. Clusters of top ranking residues in 1bccC Overlap with known functional surfaces at 28% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. PEE binding site. Table 64 lists the top 28% of residues at the interface with 1bccPEE7 (pee). The following table (Table 65) suggests Table W W(100) / R R(100) / Y Y(100) / Fig. 53. Residues in 1bccC, at the interface with PEE, colored by their relative importance. The ligand (PEE) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccC.) 34

35 Figure 53 shows residues in 1bccC colored by their importance, at the interface with 1bccPEE7. Ubiquinone-10 binding site. Table 66 lists the top 28% of residues at the interface with 1bccU105 (ubiquinone-10). The following table (Table 67) suggests possible disruptive replacements for these residues (see Section Table H H(100) / Table 66. The top 28% of residues in 1bccC at the interface with ubiquinone-10.(field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table H (E)(TQMD)(SNKVCLAPIG)(YR) Table 67. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with ubiquinone-10. Figure 54 shows residues in 1bccC colored by their importance, at the interface with 1bccU105. Interface with 1bccF.Table 68 lists the top 28% of residues at the interface with 1bccF. The following table (Table 69) suggests Table N N(100) / P P(100) / G G(100) / D D(100) / R R(100) / Table 68. The top 28% of residues in 1bccC at the interface with 1bccF. Table N (Y)(FTWH)(SEVCARG)(MD) 209 P (YR)(TH)(SKECG)(FQWD) 211 G (KER)(FQMWHD)(NYLPI)(SVA) 217 D (R)(FWH)(KYVCAG)(TQM) 319 R (TD)(SYEVCLAPIG)(FMW)(N) Table 69. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with 1bccF. Figure 55 shows residues in 1bccC colored by their importance, at the interface with 1bccF. Interface with 1bccE.Table 70 lists the top 28% of residues at the interface with 1bccE. The following table (Table 71) suggests Table T T(100) / R R(100) / V V(100) / G G(100) / Fig. 54. Residues in 1bccC, at the interface with ubiquinone-10, colored by their relative importance. The ligand (ubiquinone-10) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccC.) Table 70. The top 28% of residues in 1bccC at the interface with 1bccE. 35

36 Fig. 55. Residues in 1bccC, at the interface with 1bccF, colored by their relative importance. 1bccF is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccC.) Fig. 56. Residues in 1bccC, at the interface with 1bccE, colored by their relative importance. 1bccE is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccC.) Table T (KR)(FQMWH)(NELPI)(D) 72 R (TD)(SYEVCLAPIG)(FMW)(N) 74 V (KYER)(QHD)(N)(FTMW) 77 G (KER)(FQMWHD)(NYLPI)(SVA) Table 71. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with 1bccE. Figure 56 shows residues in 1bccC colored by their importance, at the interface with 1bccE. Interface with 1bccD.Table 72 lists the top 28% of residues at the interface with 1bccD. The following table (Table 73) suggests Table F F(100) / R R(100) / W W(100) / D D(100) / P P(100) / P P(100) / Table 72. The top 28% of residues in 1bccC at the interface with 1bccD. Table F (KE)(TQD)(SNCRG)(M) 72 R (TD)(SYEVCLAPIG)(FMW)(N) 78 W (KE)(TQD)(SNCRG)(M) 217 D (R)(FWH)(KYVCAG)(TQM) 223 P (YR)(TH)(SKECG)(FQWD) 248 P (YR)(TH)(SKECG)(FQWD) Table 73. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with 1bccD. Figure 57 shows residues in 1bccC colored by their importance, at the interface with 1bccD. HEM binding site. Table 74 lists the top 28% of residues at the interface with 1bccHEM1 (hem). The following table (Table 75) suggests Table 74. antn 49 G G(100) / L L(100) / Y Y(100) /

37 Table 75. continued 91 F (KE)(TQD)(SNCRG)(M) 131 G (KER)(FQMWHD)(NYLPI)(SVA) 132 Y (K)(QM)(NEVLAPIR)(D) 134 L (YR)(TH)(SKECG)(FQWD) 180 F (KE)(TQD)(SNCRG)(M) 183 H (E)(TQMD)(SNKVCLAPIG)(YR) 187 P (YR)(TH)(SKECG)(FQWD) 274 Y (K)(QM)(NEVLAPIR)(D) Table 75. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with HEM. Fig. 57. Residues in 1bccC, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccC.) Table 74. continued antn 84 H H(100) / site 88 A A(100) / F F(100) / G G(100) / Y Y(100) / L L(100) / F F(100) / H H(100) / site 187 P P(100) / Y Y(100) / Table 74. The top 28% of residues in 1bccC at the interface with HEM.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table G (KER)(FQMWHD)(NYLPI)(SVA) 52 L (YR)(TH)(SKECG)(FQWD) 56 Y (K)(QM)(NEVLAPIR)(D) 84 H (E)(TQMD)(SNKVCLAPIG)(YR) 88 A (KYER)(QHD)(N)(FTMW) Fig. 58. Residues in 1bccC, at the interface with HEM, colored by their relative importance. The ligand (HEM) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccC.) Figure 58 shows residues in 1bccC colored by their importance, at the interface with 1bccHEM1. HEM binding site. Table 76 lists the top 28% of residues at the interface with 1bccHEM2 (hem). The following table (Table 77) suggests Table 76. antn 31 W W(100) / F F(100) /

38 Table 76. continued antn 35 G G(100) / F F(100) / H H(100) / site 101 R R(100) / S S(100) / Y Y(100) / W W(100) / G G(100) / H H(100) / site 208 N N(100) / Table 76. The top 28% of residues in 1bccC at the interface with HEM.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table W (KE)(TQD)(SNCRG)(M) 34 F (KE)(TQD)(SNCRG)(M) 35 G (KER)(FQMWHD)(NYLPI)(SVA) 91 F (KE)(TQD)(SNCRG)(M) 98 H (E)(TQMD)(SNKVCLAPIG)(YR) 101 R (TD)(SYEVCLAPIG)(FMW)(N) 107 S (KR)(FQMWH)(NYELPI)(D) 108 Y (K)(QM)(NEVLAPIR)(D) 114 W (KE)(TQD)(SNCRG)(M) 117 G (KER)(FQMWHD)(NYLPI)(SVA) 197 H (E)(TQMD)(SNKVCLAPIG)(YR) 208 N (Y)(FTWH)(SEVCARG)(MD) Table 77. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with HEM. Figure 59 shows residues in 1bccC colored by their importance, at the interface with 1bccHEM2. Interface with 1bccG.Table 78 lists the top 28% of residues at the interface with 1bccG. The following table (Table 79) suggests Table H H(100) / Table 78. The top 28% of residues in 1bccC at the interface with 1bccG. Fig. 59. Residues in 1bccC, at the interface with HEM, colored by their relative importance. The ligand (HEM) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccC.) Table H (E)(TQMD)(SNKVCLAPIG)(YR) Table 79. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with 1bccG. Figure 60 shows residues in 1bccC colored by their importance, at the interface with 1bccG. Interface with 1bccA.Table 80 lists the top 28% of residues at the interface with 1bccA. The following table (Table 81) suggests Table P P(100) / Table 80. The top 28% of residues in 1bccC at the interface with 1bccA. 38

39 Fig. 60. Residues in 1bccC, at the interface with 1bccG, colored by their relative importance. 1bccG is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccC.) Fig. 61. Residues in 1bccC, at the interface with 1bccA, colored by their relative importance. 1bccA is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccC.) Table P (YR)(TH)(SKECG)(FQWD) Table 81. List of disruptive for the top 28% of residues in 1bccC, that are at the interface with 1bccA. Figure 61 shows residues in 1bccC colored by their importance, at the interface with 1bccA Possible novel functional surfaces at 28% coverage. One group of residues is conserved on the 1bccC surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 62. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 82, while Table 83 suggests possible disruptive replacements for these residues (see Section Table 82. res type substitutions(%) cvg antn 31 W W(100) F F(100) G G(100) H H(100) 0.20 site 101 R R(100) Y Y(100) S S(100) Y Y(100) 0.20 Fig. 62. A possible active surface on the chain 1bccC. The larger cluster it belongs to is shown in blue. Table 82. continued res type substitutions(%) cvg antn 114 W W(100) G G(100) H H(100) 0.20 site 208 N N(100) P P(100) G G(100) P P(100) L L(100) 0.20 Table 82. Residues forming surface patch in 1bccC. 39

40 Table W (KE)(TQD)(SNCRG)(M) 34 F (KE)(TQD)(SNCRG)(M) 35 G (KER)(FQMWHD)(NYLPI)(SVA) 98 H (E)(TQMD)(SNKVCLAPIG)(YR) 101 R (TD)(SYEVCLAPIG)(FMW)(N) 105 Y (K)(QM)(NEVLAPIR)(D) 107 S (KR)(FQMWH)(NYELPI)(D) 108 Y (K)(QM)(NEVLAPIR)(D) 114 W (KE)(TQD)(SNCRG)(M) 117 G (KER)(FQMWHD)(NYLPI)(SVA) 197 H (E)(TQMD)(SNKVCLAPIG)(YR) 208 N (Y)(FTWH)(SEVCARG)(MD) 209 P (YR)(TH)(SKECG)(FQWD) 211 G (KER)(FQMWHD)(NYLPI)(SVA) 306 P (YR)(TH)(SKECG)(FQWD) 308 L (YR)(TH)(SKECG)(FQWD) Table 83. Disruptive for the surface patch in 1bccC. Another group of surface residues is shown in Fig.63. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Table 84. continued res type substitutions(%) cvg antn 126 A A(100) F F(100) G G(100) Y Y(100) L L(100) M M(100) G G(100) I I(100) L L(100) A A(100) P P(100) G G(100) R R(100) F F(100) H H(100) 0.20 site 186 L L(100) P P(100) N N(100) T T(100) P P(100) I I(100) P P(100) W W(100) Y Y(100) L L(100) G G(100) 0.20 Table 84. Residues forming surface patch in 1bccC. Fig. 63. Another possible active surface on the chain 1bccC. The larger cluster it belongs to is shown in blue. The residues belonging to this surface patch are listed in Table 84, while Table 85 suggests possible disruptive replacements for these residues (see Section Table 84. res type substitutions(%) cvg antn 48 T T(100) G G(100) L L(100) Y Y(100) F F(100) H H(100) 0.20 site 87 G G(100) A A(100) F F(100) 0.20 Table T (KR)(FQMWH)(NELPI)(D) 49 G (KER)(FQMWHD)(NYLPI)(SVA) 52 L (YR)(TH)(SKECG)(FQWD) 56 Y (K)(QM)(NEVLAPIR)(D) 64 F (KE)(TQD)(SNCRG)(M) 84 H (E)(TQMD)(SNKVCLAPIG)(YR) 87 G (KER)(FQMWHD)(NYLPI)(SVA) 88 A (KYER)(QHD)(N)(FTMW) 91 F (KE)(TQD)(SNCRG)(M) 126 A (KYER)(QHD)(N)(FTMW) 129 F (KE)(TQD)(SNCRG)(M) 131 G (KER)(FQMWHD)(NYLPI)(SVA) 132 Y (K)(QM)(NEVLAPIR)(D) 134 L (YR)(TH)(SKECG)(FQWD) 139 M (Y)(TH)(SCRG)(FWD) 143 G (KER)(FQMWHD)(NYLPI)(SVA) 147 I (YR)(TH)(SKECG)(FQWD) 150 L (YR)(TH)(SKECG)(FQWD) 153 A (KYER)(QHD)(N)(FTMW) 155 P (YR)(TH)(SKECG)(FQWD) 40

41 Table 85. continued 167 G (KER)(FQMWHD)(NYLPI)(SVA) 178 R (TD)(SYEVCLAPIG)(FMW)(N) 180 F (KE)(TQD)(SNCRG)(M) 183 H (E)(TQMD)(SNKVCLAPIG)(YR) 186 L (YR)(TH)(SKECG)(FQWD) 187 P (YR)(TH)(SKECG)(FQWD) 256 N (Y)(FTWH)(SEVCARG)(MD) 265 T (KR)(FQMWH)(NELPI)(D) 266 P (YR)(TH)(SKECG)(FQWD) 269 I (YR)(TH)(SKECG)(FQWD) 271 P (YR)(TH)(SKECG)(FQWD) 273 W (KE)(TQD)(SNCRG)(M) 274 Y (K)(QM)(NEVLAPIR)(D) 289 L (YR)(TH)(SKECG)(FQWD) 291 G (KER)(FQMWHD)(NYLPI)(SVA) Fig. 64. Residues in 1bccD colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Table 85. Disruptive for the surface patch in 1bccC. Fig. 65. Residues in 1bccD colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) 9 CHAIN 1BCCD 9.1 P00125 overview From SwissProt, id P00125, 89% identical to 1bccD: Description: Cytochrome c1, heme protein, mitochondrial (Cytochrome c-1). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is the heme-containing component of the cytochrome b-c1 complex, which accepts electrons from Rieske protein and transfers electrons to cytochrome c in the mitochondrial respiratory chain. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial intermembrane space. Ptm: Binds 1 heme group per subunit. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed. 9.2 Multiple sequence alignment for 1bccD For the chain 1bccD, the alignment 1bccD.msf (attached) with 171 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccD.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 171 Total number of residues: Smallest: 185 Largest: 241 Average length: Alignment length: 241 Average identity: 44% Most related pair: 99% Most unrelated pair: 18% Most distant seq: 35% Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 21% eukaryotic ( 2% vertebrata, 2% arthropoda, 7% fungi, 3% plantae), and 12% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccD.descr. 9.3 Residue ranking in 1bccD The 1bccD sequence is shown in Figs , with each residue colored according to its estimated importance. The full listing of residues in 1bccD can be found in the file called 1bccD.ranks sorted in the attachment. 9.4 Top ranking residues in 1bccD and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein. Figure 66 shows residues in 1bccD colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment Clustering of residues at 25% coverage. Fig. 67 shows the top 25% of all residues, this time colored according to clusters they 41

42 Table 86. cluster size member color residues red 53 12,14,16,22,25,26,27,28,29 32,33,35,36,37,40,41,94,97 101,105,107,110,111,112, ,115,118,119,120,123, ,131,133,134,150,153, ,174,181,185,186,189, ,194,195,196,201,202,205 blue 2 83,89 yellow 2 49,51 green 2 223,227 Table 86. Clusters of top ranking residues in 1bccD. Fig. 66. Residues in 1bccD, colored by their relative importance. Clockwise: front, back, top and bottom views. belong to. The clusters in Fig.67 are composed of the residues listed Fig. 67. Residues in 1bccD, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. in Table Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. HEM binding site. Table 87 lists the top 25% of residues at the interface with 1bccHEM3 (hem). The following table (Table 88) suggests Table 87. antn 40 C C(100) / H H(100) / site 37 C C(97) / F(2) 120 R R(96) / K(2)HL 110 P A(14) / P(83) G(1) 126 Y Y(93) / W(5)HE 111 P P(97)TL / V(1) 190 L L(94) / I(2) M(3) 36 V V(89) / I(3) Y(5)NDL 160 M M(97) / site A(1) G(1)I 113 L L(87) / M(5) F(6) 32 V V(85) / I(9) 42

43 Table 87. continued antn L(4)Y 186 V V(88) / I(8) L(2)T 130 L L(83) / V(2) Y(5) I(7)F 153 F F(87) / Y(2) M(5) G(1) A(1)WVL 105 N S(9) / N(79).(4) V(1) W(1)QFA H 131 L M(7) / L(88) I(2) V(2) Table 88. continued Table 88. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with HEM. Table 87. The top 25% of residues in 1bccD at the interface with HEM.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Table C (KER)(FQMWHD)(NYLPI)(SVA) 41 H (E)(TQMD)(SNKVCLAPIG)(YR) 37 C (KE)(R)(QD)(M) 120 R (T)(D)(Y)(SECG) 110 P (R)(Y)(H)(KE) 126 Y (K)(Q)(M)(NEVAR) 111 P (R)(Y)(H)(K) 190 L (Y)(R)(TH)(CG) 36 V (R)(K)(Y)(E) 160 M (Y)(H)(R)(T) 113 L (YR)(T)(H)(KECG) 32 V (R)(K)(E)(Y) 186 V (R)(KY)(E)(H) 130 L (R)(Y)(K)(E) 153 F (K)(E)(QD)(R) 105 N (Y)(T)(E)(H) 131 L (Y)(R)(H)(T) Fig. 68. Residues in 1bccD, at the interface with HEM, colored by their relative importance. The ligand (HEM) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccD.) Figure 68 shows residues in 1bccD colored by their importance, at the interface with 1bccHEM3. Interface with 1bccF.Table 89 lists the top 25% of residues at the interface with 1bccF. The following table (Table 90) suggests Table W W(94) / Y(4)SR Table 89. The top 25% of residues in 1bccD at the interface with 1bccF. 43

44 Table W (KE)(QD)(T)(N) Table 90. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccF. Fig. 69. Residues in 1bccD, at the interface with 1bccF, colored by their relative importance. 1bccF is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Figure 69 shows residues in 1bccD colored by their importance, at the interface with 1bccF. Interface with 1bccC.Table 91 lists the top 25% of residues at the interface with 1bccC. The following table (Table 92) suggests Table R R(99)M / R R(96) / K(2)HL 196 P P(96) / S(2)RY 227 W W(94) / Y(4)SR 49 R R(90) / S(2)E T(2) Table 91. continued Q(1) D(1) N(1)G 195 E E(92) / D(4)YNM RH 114 S S(93) / T(4) Q(2) 205 G G(90) / R(1) L(2) F(2) T(1)C A(1) 119 A A(83) / K(2) V(2) G(9) S(1)LRT 118 R K(73) / T(1) A(1) R(9) S(5)G N(2) L(3) F(2) 115 Y L(78) / V(7) Y(8) M(1) H(2)KSC R 202 K K(85) / H(5) R(5) Q(1)VP 223 K N(22) / K(58) Q(2) Y(8) M(1)W R(4)CTF 194 A A(80) / G(1) S(16)TQ Table 91. The top 25% of residues in 1bccD at the interface with 1bccC. 44

45 Table R (T)(YD)(SCG)(EVA) 120 R (T)(D)(Y)(SECG) 196 P (R)(Y)(TKEH)(CG) 227 W (KE)(QD)(T)(N) 49 R (Y)(FTW)(D)(VA) 195 E (FW)(H)(VCAG)(Y) 114 S (R)(FWH)(K)(M) 205 G (E)(K)(R)(D) 119 A (Y)(E)(R)(K) 118 R (D)(YE)(T)(SFCLWPIG) 115 Y (K)(Q)(E)(M) 202 K (Y)(T)(FW)(SCG) 223 K (Y)(T)(FW)(S) 194 A (R)(K)(YE)(H) Table 92. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccC. Fig. 70. Residues in 1bccD, at the interface with 1bccC, colored by their relative importance. 1bccC is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Figure 70 shows residues in 1bccD colored by their importance, at the interface with 1bccC. Interface with 1bccJ.Table 93 lists the top 25% of residues at the interface with 1bccJ. The following table (Table 94) suggests possible disruptive replacements for these residues (see Section 45

46 Table D D(94) / N(4)SA 27 R Q(53) / R(43) K(2) 14 H F(50) / H(35) S(2).(5) Q(1)Y W(1)K 202 K K(85) / H(5) R(5) Q(1)VP Table 93. The top 25% of residues in 1bccD at the interface with 1bccJ. Fig. 71. Residues in 1bccD, at the interface with 1bccJ, colored by their relative importance. 1bccJ is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Table D (R)(H)(FW)(Y) 27 R (T)(Y)(D)(SVCAG) 14 H (E)(TD)(QM)(KCG) 202 K (Y)(T)(FW)(SCG) Table 94. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccJ. Figure 71 shows residues in 1bccD colored by their importance, at the interface with 1bccJ. Interface with 1bccE.Table 95 lists the top 25% of residues at the interface with 1bccE. The following table (Table 96) suggests Table R R(90) / S(2)E T(2) Q(1) D(1) N(1)G Table 95. The top 25% of residues in 1bccD at the interface with 1bccE. Table R (Y)(FTW)(D)(VA) Table 96. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccE. Figure 72 shows residues in 1bccD colored by their importance, at the interface with 1bccE. Interface with 1bccA.Table 97 lists the top 25% of residues at the interface with 1bccA. The following table (Table 98) suggests Table W W(94) / Y(4)SR Table 97. The top 25% of residues in 1bccD at the interface with 1bccA. 46

47 Table W (KE)(QD)(T)(N) Table 98. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccA. Fig. 72. Residues in 1bccD, at the interface with 1bccE, colored by their relative importance. 1bccE is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Fig. 73. Residues in 1bccD, at the interface with 1bccA, colored by their relative importance. 1bccA is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Figure 73 shows residues in 1bccD colored by their importance, at the interface with 1bccA. Interface with 1bccH.Table 99 lists the top 25% of residues at the interface with 1bccH. The following table (Table 100) suggests Table D D(100) / R R(99)T / D D(92) / Q(3) G(2) K(1)A 12 W F(23) / W(67).(6) L(1) 47

48 Table 99. continued 133 G G(88) / C(1) S(5) T(2)KHA F 181 Q N(5) / Q(83) H(5) S(1)E A(3)R 138 P E(10) / P(80) S(2) T(1) D(4) K(1) Table 99. The top 25% of residues in 1bccD at the interface with 1bccH. Fig. 74. Residues in 1bccD, at the interface with 1bccH, colored by their relative importance. 1bccH is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccD.) Table D (R)(FWH)(KYVCAG)(TQM) 28 R (D)(ELPI)(FTYVMAW)(SCG) 173 D (R)(FWH)(Y)(KVCAG) 12 W (KE)(T)(QD)(SCRG) 133 G (E)(K)(R)(QD) 181 Q (Y)(FW)(T)(H) 138 P (R)(Y)(H)(T) Table 100. List of disruptive for the top 25% of residues in 1bccD, that are at the interface with 1bccH. Figure 74 shows residues in 1bccD colored by their importance, at the interface with 1bccH Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1bccD surface, away from (or susbtantially larger than) other functional sites and interfaces recognizable in PDB entry 1bcc. It is shown in Fig. 75. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface patch are listed in Table 101, while Table 102 suggests possible disruptive replacements for these residues (see Section Table 101. res type substitutions(%) cvg antn 40 C C(100) 0.02 Fig. 75. A possible active surface on the chain 1bccD. The larger cluster it belongs to is shown in blue. Table 101. continued res type substitutions(%) cvg antn 41 H H(100) 0.02 site 112 D D(100) R R(99)T C C(97)F(2) R R(99)M R R(96)K(2)HL P P(96)S(2)RY P A(14)P(83)G(1) Y Y(93)W(5)HE W W(94)Y(4)F(1)G P P(97)TLV(1) G G(97)SHAY L L(94)I(2)M(3) D D(94)N(4)SA

49 Table 101. continued res type substitutions(%) cvg antn 36 V V(89)I(3)Y(5)ND 0.08 L 33 Y Y(91)F(8)A R Q(53)R(43)K(2) E E(92)D(4)YNMRH M M(97)A(1)G(1)I 0.11 site 173 D D(92)Q(3)G(2) 0.11 K(1)A 12 W F(23)W(67).(6) 0.12 L(1) 113 L L(87)M(5)F(6) Y Y(88)F(11) H F(50)H(35)S(2) 0.14.(5)Q(1)YW(1)K 133 G G(88)C(1)S(5) 0.14 T(2)KHAF 205 G G(90)R(1)L(2) 0.14 F(2)T(1)CA(1) 16 G G(76)S(2)Q(2) 0.15.(5)FN(1)LK(5) D(2)RA 32 V V(85)I(9)L(4)Y V V(88)I(8)L(2)T P.(18)P(76)VA(1) 0.17 S(1)QTR 107 G L(7)G(80)N(3) 0.17 A(2)E(1)S(1).(1)IT 130 L L(83)V(2)Y(5) 0.17 I(7)F 119 A A(83)K(2)V(2) 0.18 G(9)S(1)LRT 101 A.(16)A(76)LV 0.19 S(1)KERD(1)PG 118 R K(73)T(1)A(1) 0.19 R(9)S(5)GN(2) L(3)F(2) 97 N.(17)N(76)T(1) 0.20 M(1)ASPD(1)K 115 Y L(78)V(7)Y(8) 0.20 M(1)H(2)KSCR 181 Q N(5)Q(83)H(5) 0.20 S(1)EA(3)R 202 K K(85)H(5)R(5) 0.21 Q(1)VP 35 Q E(63)Q(26)KN(5) 0.22 D(1)A(1)R 153 F F(87)Y(2)M(5) 0.22 G(1)A(1)WVL 105 N S(9)N(79).(4) 0.23 V(1)W(1)QFAH 131 L M(7)L(88)I(2) 0.23 Table 101. continued res type substitutions(%) cvg antn V(2) 174 G G(89)E(2)Q(1) 0.24 A(1)PD(1)IN(1) L(1) Table 101. Residues forming surface patch in 1bccD. Table C (KER)(FQMWHD)(NYLPI)(SVA) 41 H (E)(TQMD)(SNKVCLAPIG)(YR) 112 D (R)(FWH)(KYVCAG)(TQM) 28 R (D)(ELPI)(FTYVMAW)(SCG) 37 C (KE)(R)(QD)(M) 201 R (T)(YD)(SCG)(EVA) 120 R (T)(D)(Y)(SECG) 196 P (R)(Y)(TKEH)(CG) 110 P (R)(Y)(H)(KE) 126 Y (K)(Q)(M)(NEVAR) 192 W (K)(E)(Q)(D) 111 P (R)(Y)(H)(K) 123 G (K)(E)(R)(Q) 190 L (Y)(R)(TH)(CG) 22 D (R)(H)(FW)(Y) 36 V (R)(K)(Y)(E) 33 Y (K)(Q)(E)(R) 27 R (T)(Y)(D)(SVCAG) 195 E (FW)(H)(VCAG)(Y) 160 M (Y)(H)(R)(T) 173 D (R)(FWH)(Y)(KVCAG) 12 W (KE)(T)(QD)(SCRG) 113 L (YR)(T)(H)(KECG) 134 Y (K)(Q)(EM)(NR) 14 H (E)(TD)(QM)(KCG) 133 G (E)(K)(R)(QD) 205 G (E)(K)(R)(D) 16 G (R)(E)(K)(H) 32 V (R)(K)(E)(Y) 186 V (R)(KY)(E)(H) 94 P (Y)(R)(H)(TE) 107 G (R)(K)(H)(E) 130 L (R)(Y)(K)(E) 119 A (Y)(E)(R)(K) 101 A (Y)(R)(K)(E) 118 R (D)(YE)(T)(SFCLWPIG) 97 N (Y)(H)(FW)(R) 115 Y (K)(Q)(E)(M) 181 Q (Y)(FW)(T)(H) 202 K (Y)(T)(FW)(SCG) 49

50 Table 102. continued 35 Q (Y)(FW)(H)(T) 153 F (K)(E)(QD)(R) 105 N (Y)(T)(E)(H) 131 L (Y)(R)(H)(T) 174 G (R)(H)(K)(Y) Fig. 76. Residues 2-79 in 1bccG colored by their relative importance. (See Appendix, Fig.89, for the coloring scheme.) Table 102. Disruptive for the surface patch in 1bccD. 10 CHAIN 1BCCG 10.1 P13271 overview From SwissProt, id P13271, 89% identical to 1bccG: Description: Ubiquinol-cytochrome c reductase complex ubiquinone-binding protein QP-C (EC ) (Ubiquinolcytochrome c reductase complex 9.5 kda protein) (Complex III subunit VII). Organism, scientific name: Bos taurus (Bovine). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Bovinae; Bos. Function: This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This subunit, together with cytochrome b, binds to ubiquinone. Catalytic activity: QH(2) + 2 ferricytochrome c = Q + 2 ferrocytochrome c. Subunit: The bc1 complex contains 11 subunits; 3 respiratory subunits (cytochrome b, cytochrome c1, Rieske protein), 2 core proteins and 6 low-molecular weight proteins. Subcellular location: Mitochondrial inner membrane. Similarity: Belongs to the UQCRQ/QCR8 family. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed Multiple sequence alignment for 1bccG For the chain 1bccG, the alignment 1bccG.msf (attached) with 32 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1bccG.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 32 Total number of residues: 2373 Smallest: 59 Largest: 78 Average length: 74.2 Alignment length: 78 Average identity: 48% Most related pair: 99% Most unrelated pair: 25% Most distant seq: 53% Furthermore, 6% of residues show as conserved in this alignment. The alignment consists of 43% eukaryotic ( 21% vertebrata, 12% arthropoda, 9% fungi) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1bccG.descr Residue ranking in 1bccG The 1bccG sequence is shown in Fig. 76, with each residue colored according to its estimated importance. The full listing of residues in 1bccG can be found in the file called 1bccG.ranks sorted in the attachment Top ranking residues in 1bccG and their position on the structure In the following we consider residues ranking among top 26% of residues in the protein (the closest this analysis allows us to get to 25%). Figure 77 shows residues in 1bccG colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment Clustering of residues at 26% coverage. Fig. 78 shows the top 26% of all residues, this time colored according to clusters they belong to. The clusters in Fig.78 are composed of the residues listed in Table 103. Table 103. cluster size member color residues red 6 48,50,51,52,56,59 blue 5 33,35,36,39,40 yellow 3 19,22,23 green 2 25,27 purple 2 71,72 Table 103. Clusters of top ranking residues in 1bccG. 50

51 PEE binding site. Table 104 lists the top 26% of residues at the interface with 1bccPEE7 (pee). The following table (Table 105) suggests Table R R(100) / F F(93) / M(3) L(3) 48 V I(12) / V(87) Table 104. The top 26% of residues in 1bccG at the interface with PEE.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance Fig. 77. Residues in 1bccG, colored by their relative importance. Clockwise: front, back, top and bottom views. Table R (TD)(SYEVCLAPIG)(FMW)(N) 52 F (T)(KE)(DR)(QCG) 48 V (YR)(KE)(H)(QD) Table 105. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with PEE. Figure 79 shows residues in 1bccG colored by their importance, at the interface with 1bccPEE7. Interface with 1bccD.Table 106 lists the top 26% of residues at the interface with 1bccD. The following table (Table 107) suggests Fig. 78. Residues in 1bccG, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table Q Q(100) / P P(40) / A(59) 27 P A(71) / P(28) 16 Y Y(87) / H(9) F(3) 19 S T(3) / S(90) A(6) Overlap with known functional surfaces at 26% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Table 106. The top 26% of residues in 1bccG at the interface with 1bccD. 51

52 Fig. 79. Residues in 1bccG, at the interface with PEE, colored by their relative importance. The ligand (PEE) is colored green. Atoms further than 30Å away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1bccG.) Table Q (Y)(FTWH)(SVCAG)(D) 25 P (YR)(H)(TKE)(SQCDG) 27 P (YR)(H)(TKE)(SQCDG) 16 Y (K)(Q)(EM)(N) 19 S (KR)(QH)(FMW)(E) Table 107. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with 1bccD. Figure 80 shows residues in 1bccG colored by their importance, at the interface with 1bccD. Interface with 1bccF.Table 108 lists the top 26% of residues at the interface with 1bccF. The following table (Table 109) suggests Table N N(100) / R R(100) / Fig. 80. Residues in 1bccG, at the interface with 1bccD, colored by their relative importance. 1bccD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccG.) Table 108. continued 40 R R(100) / Table 108. The top 26% of residues in 1bccG at the interface with 1bccF. Table N (Y)(FTWH)(SEVCARG)(MD) 39 R (TD)(SYEVCLAPIG)(FMW)(N) 40 R (TD)(SYEVCLAPIG)(FMW)(N) Table 109. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with 1bccF. Figure 81 shows residues in 1bccG colored by their importance, at the interface with 1bccF. Interface with 1bccC.Table 110 lists the top 26% of residues at the interface with 1bccC. The following table (Table 111) suggests 52

53 Fig. 81. Residues in 1bccG, at the interface with 1bccF, colored by their relative importance. 1bccF is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccG.) Fig. 82. Residues in 1bccG, at the interface with 1bccC, colored by their relative importance. 1bccC is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccG.) Table Y M(34) / Y(65) 51 P P(93) / Q(6) 52 F F(93) / M(3) L(3) 48 V I(12) / V(87) Table 110. The top 26% of residues in 1bccG at the interface with 1bccC. Table Y (K)(QR)(NEVMA)(LPDI) 51 P (Y)(THR)(SCG)(FEW) 52 F (T)(KE)(DR)(QCG) 48 V (YR)(KE)(H)(QD) Figure 82 shows residues in 1bccG colored by their importance, at the interface with 1bccC. Interface with 1bccE.Table 112 lists the top 26% of residues at the interface with 1bccE. The following table (Table 113) suggests Table Q Q(100) / E R(34) / E(62) Q(3) 25 P P(40) / A(59) 27 P A(71) / P(28) 16 Y Y(87) / H(9) F(3) Table 112. The top 26% of residues in 1bccG at the interface with 1bccE. Table 111. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with 1bccC. 53

54 Table Q (Y)(FTWH)(SVCAG)(D) 22 E (FW)(YH)(VCAG)(T) 25 P (YR)(H)(TKE)(SQCDG) 27 P (YR)(H)(TKE)(SQCDG) 16 Y (K)(Q)(EM)(N) Table 113. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with 1bccE. Table K (Y)(T)(FW)(VCAG) Table 115. List of disruptive for the top 26% of residues in 1bccG, that are at the interface with 1bccH. Fig. 83. Residues in 1bccG, at the interface with 1bccE, colored by their relative importance. 1bccE is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccG.) Figure 83 shows residues in 1bccG colored by their importance, at the interface with 1bccE. Interface with 1bccH.Table 114 lists the top 26% of residues at the interface with 1bccH. The following table (Table 115) suggests Table K K(93) / (3) R(3) Table 114. The top 26% of residues in 1bccG at the interface with 1bccH. Fig. 84. Residues in 1bccG, at the interface with 1bccH, colored by their relative importance. 1bccH is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1bccG.) Figure 84 shows residues in 1bccG colored by their importance, at the interface with 1bccH. Interface with 1bccA.Table 116 lists the top 26% of residues at the interface with 1bccA. The following table (Table 117) suggests Table E R(34) / E(62) Q(3) 5 G.(9) / G(90) 16 Y Y(87) /