Pages 1–16 2blf Evolutionary trace report by report maker August 29, 2009

4 Notes on using trace results 13 4.1 Coverage 13 4.2 Known substitutions 14 4.3 Surface 14 4.4 Number of contacts 14 4.5 Annotation 14 4.6 Mutation suggestions 14

5 Appendix 14 5.1 File formats 14 5.2 Color schemes used 14 5.3 Credits 15 5.3.1 Alistat 15 5.3.2 CE 15 5.3.3 DSSP 15 5.3.4 HSSP 15 5.3.5 LaTex 15 5.3.6 Muscle 15 5.3.7 Pymol 15 5.4 Note about ET Viewer 15 5.5 Citing this work 15 5.6 About report maker 15 CONTENTS 5.7 Attachments 15

1 Introduction 1 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2blf): 2 Chain 2blfA 1 Title: Sulfite dehydrogenase from starkeya novella 2.1 Q9LA16 overview 1 Compound: Mol id: 1; molecule: sulfite:cytochrome c oxidoreduc- 2.2 Multiple sequence alignment for 2blfA 1 tase subunit a; chain: a; synonym: sora; engineered: yes; mol id: 2; 2.3 Residue ranking in 2blfA 1 molecule: sulfite:cytochrome c oxidoreductase subunit b; chain: b; 2.4 Top ranking residues in 2blfA and their position on synonym: sorb; engineered: yes the structure 1 Organism, scientific name: Thiobacillus Novellus; 2.4.1 Clustering of residues at 25% coverage. 1 2blf contains unique chains 2blfA (373 residues) and 2blfB (81 2.4.2 Overlap with known functional surfaces at residues) 25% coverage. 2 2.4.3 Possible novel functional surfaces at 25% 2 CHAIN 2BLFA coverage. 6 2.1 Q9LA16 overview 3 Chain 2blfB 9 From SwissProt, id Q9LA16, 100% identical to 2blfA: 3.1 Q9LA15 overview 9 Description: Sulfite:cytochrome c oxidoreductase subunit A. 3.2 Multiple sequence alignment for 2blfB 9 Organism, scientific name: Thiobacillus novellus. 3.3 Residue ranking in 2blfB 10 Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Rhizo- 3.4 Top ranking residues in 2blfB and their position on biales; Hyphomicrobiaceae; Starkeya. the structure 10 3.4.1 Clustering of residues at 25% coverage. 10 2.2 Multiple sequence alignment for 2blfA 3.4.2 Overlap with known functional surfaces at For the chain 2blfA, the alignment 2blfA.msf (attached) with 226 25% coverage. 11 sequences was used. The alignment was downloaded from the HSSP 3.4.3 Possible novel functional surfaces at 25% database, and fragments shorter than 75% of the query as well as coverage. 12 duplicate sequences were removed. It can be found in the attachment

1 Lichtarge lab 2006 2.4 Top ranking residues in 2blfA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 2blfA 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. 1. Residues 1-186 in 2blfA colored by their relative importance. (See Appendix, Fig.18, for the coloring scheme.)

Fig. 2. Residues 187-373 in 2blfA colored by their relative importance. (See Appendix, Fig.18, for the coloring scheme.)

to this report, under the name of 2blfA.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 2blfA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 226 Total number of residues: 78267 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 280 top 25% of all residues, this time colored according to clusters they Largest: 373 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 346.3 in Table 1. Alignment length: 373 Average identity: 30% Table 1. Most related pair: 98% cluster size member Most unrelated pair: 16% color residues Most distant seq: 31% red 93 47,48,53,54,55,56,57,70,72 76,78,89,103,104,105,106,107 108,109,118,120,121,125,127 Furthermore, <1% of residues show as conserved in this ali- 128,129,131,133,134,136,140 gnment. 143,144,156,158,170,184,185 The alignment consists of 8% eukaryotic ( 3% vertebrata, <1% 186,188,189,190,193,194,197 arthropoda, 2% fungi, 1% plantae), 9% prokaryotic, and <1% 198,200,202,205,206,207,209 archaean sequences. (Descriptions of some sequences were not rea- 210,211,212,213,214,215,216 dily available.) The file containing the sequence descriptions can be 217,220,231,232,235,236,237 found in the attachment, under the name 2blfA.descr. 265,267,285,286,287,288,289 290,297,300,302,304,307,310 2.3 Residue ranking in 2blfA 321,324,339,340,341,343,346 349,350,355,356,359,360 The 2blfA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 2blfA can be found in the file called 2blfA.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) P(2) Q(1) S(3)IEK 56 Y S(4) 0.20 2/2 3.95 site N(44) W(7)D T(1) H(15) Y(11) V(1) I(7). A(1)MCQ 231 W F(14) 0.23 6/0 4.06 Y(9) .(4) I(5) W(30) K(7) E(12) Fig. 4. Residues in 2blfA, colored according to the cluster they belong to: V(3)T red, followed by blue and yellow are the largest clusters (see Appendix for N(1) the coloring scheme). Clockwise: front, back, top and bottom views. The Q(2) corresponding Pymol script is attached. A(3) G(1) D(2)M Table 1. continued 120 Q P(13) 0.24 34/1 3.11 cluster size member G(7) color residues S(3) Q(46) Table 1. Clusters of top ranking residues in 2blfA. .(10)M K(1) A(2) 2.4.2 Overlap with known functional surfaces at 25% coverage. T(1) The name of the ligand is composed of the source PDB identifier E(3) and the heteroatom name used in that file. N(4) Interface with 2blfB.Table 2 lists the top 25% of residues at the D(3)L interface with 2blfB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 4.6). Table 2. The top 25% of residues in 2blfA at the interface with 2blfB. Table 2. (Field names: res: residue number in the PDB entry; type: amino acid type; res type subst’s cvg noc/ dist antn substs: substitutions seen in the alignment; with the percentage of each type (%) bb (A˚ ) in the bracket; noc/bb: number of contacts with the ligand, with the number of 55 R V(2) 0.04 1/0 4.61 site contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) R(92). L(2)AGT IE Table 3. 57 H H(72) 0.05 3/3 4.22 res type disruptive N(15)T mutations D(8).CR 55 R (Y)(D)(T)(E) YF 57 H (E)(M)(Q)(D) 118 G .(12) 0.09 12/12 2.96 118 G (R)(H)(FW)(K) G(74) 56 Y (K)(QR)(M)(E) T(3) 231 W (K)(E)(T)(R) continued in next column continued in next column

3 Table 3. continued Table 4. continued res type disruptive res type subst’s cvg noc/ dist antn mutations (%) bb (A˚ ) 120 Q (Y)(H)(FW)(T) L(2)AGT IE Table 3. List of disruptive mutations for the top 25% of residues in 2blfA, 106 G G(79) 0.04 1/1 4.96 that are at the interface with 2blfB. S(7) N(3) A(8)HD. 210 G G(86) 0.04 30/30 3.22 site A(11) S(2) 57 H H(72) 0.05 30/7 2.90 N(15)T D(8).CR YF 236 Y Y(91) 0.06 11/0 3.34 site N(3) W(3).S 197 N H(68) 0.09 18/6 2.78 site N(22) Q(7) R(1)Y 216 H W(68) 0.09 2/1 4.65 Y(5) M(4)R H(11) K(5) C(2)ATS Q 105 S S(31) 0.12 10/6 2.74 site A(43) Fig. 5. Residues in 2blfA, at the interface with 2blfB, colored by their relative G(12) importance. 2blfB is shown in backbone representation (See Appendix for the V(7) coloring scheme for the protein chain 2blfA.) D(2) T(1)I. Figure 5 shows residues in 2blfA colored by their importance, at the 156 G G(75) 0.12 2/2 4.57 interface with 2blfB. A(3)D MSS binding site. Table 4 lists the top 25% of residues at the S(15) interface with 2blfAMSS1374 (mss). The following table (Table N(2)FC. 5) suggests possible disruptive replacements for these residues (see QE Section 4.6). 53 F F(63) 0.13 28/10 2.69 site Y(34).L Table 4. AV res type subst’s cvg noc/ dist antn 211 T V(9) 0.14 34/26 3.11 site (%) bb (A˚ ) G(6) 202 R R(96) 0.02 19/0 2.72 T(18) K(2)V S(9) 104 C C(96) 0.03 15/8 2.79 site M(6) V(2)QG. F(2) A A(23) 215 K K(94) 0.03 65/18 2.63 N(13) T(3) Y(7)D C(1) I(2)E 55 R V(2) 0.04 52/29 2.88 site 103 Q E(34) 0.15 3/3 4.68 R(92). continued in next column continued in next column

4 Table 4. continued Table 4. continued res type subst’s cvg noc/ dist antn res type subst’s cvg noc/ dist antn (%) bb (A˚ ) (%) bb (A˚ ) V(3) Q(4) Q(42) A(7) T(4) M(1)L C(4) A(5) Table 4. The top 25% of residues in 2blfA at the interface with M(3)GS. MSS.(Field names: res: residue number in the PDB entry; type: amino acid 158 D D(81)T 0.16 15/0 2.81 type; substs: substitutions seen in the alignment; with the percentage of each K(1) type in the bracket; noc/bb: number of contacts with the ligand, with the num- E(7)A ber of contacts realized through backbone atoms given in the bracket; dist: G(3)CL distance of closest apporach to the ligand. ) Q(1).VH 209 Y Y(31) 0.18 2/1 4.70 Table 5. I(13) F(7) res type disruptive P(7) mutations A(4) 202 R (TYD)(E)(SCG)(FVLAWPI) V(13) 104 C (E)(R)(K)(H) E(13) 215 K (FW)(Y)(H)(VA) T(3)L 55 R (Y)(D)(T)(E) S(2) 106 G (R)(K)(E)(M) Q(1)WD 210 G (KR)(E)(QH)(FMW) 56 Y S(4) 0.20 22/10 3.42 site 57 H (E)(M)(Q)(D) N(44) 236 Y (K)(QM)(E)(R) W(7)D 197 N (TY)(FVAW)(E)(SCG) T(1) 216 H (E)(D)(M)(TQ) H(15) 105 S (R)(K)(H)(Q) Y(11) 156 G (R)(K)(H)(E) V(1) 53 F (K)(E)(Q)(D) I(7). 211 T (R)(K)(H)(Q) A(1)MCQ 103 Q (Y)(H)(FW)(T) 213 W W(34) 0.20 20/16 2.91 site 158 D (R)(H)(FW)(Y) S(38) 209 Y (K)(R)(Q)(EM) N(15) 56 Y (K)(QR)(M)(E) Q(1) 213 W (E)(K)(D)(T) A(4) 54 V (R)(Y)(K)(E) C(1) 214 V (R)(Y)(KE)(H) H(2) M(1)F Table 5. List of disruptive mutations for the top 25% of residues in 2blfA, 54 V AV(48) 0.21 13/10 3.28 that are at the interface with MSS. I(9)R K(5) Figure 6 shows residues in 2blfA colored by their importance, at the E(14) interface with 2blfAMSS1374. Y(9)C Sulfate ion binding site. Table 6 lists the top 25% of residues at L(3) the interface with 2blfASO41375 (sulfate ion). The following table T(2). (Table 7) suggests possible disruptive replacements for these residues D(1)GSN (see Section 4.6). M Table 6. 214 V V(63) 0.22 22/16 3.61 res type subst’s cvg noc/ dist I(14) A˚ G(2)P (%) bb ( ) C(2) 231 W F(14) 0.23 1/1 4.89 T(2) Y(9) .(4) continued in next column I(5) continued in next column

5 Fig. 6. Residues in 2blfA, at the interface with MSS, colored by their relative Fig. 7. Residues in 2blfA, at the interface with sulfate ion, colored by their importance. The ligand (MSS) is colored green. Atoms further than 30A˚ away relative importance. The ligand (sulfate ion) is colored green. Atoms further from the geometric center of the ligand, as well as on the line of sight to the than 30A˚ away from the geometric center of the ligand, as well as on the line ligand were removed. (See Appendix for the coloring scheme for the protein of sight to the ligand were removed. (See Appendix for the coloring scheme chain 2blfA.) for the protein chain 2blfA.)

Table 6. continued Figure 7 shows residues in 2blfA colored by their importance, at the res type subst’s cvg noc/ dist interface with 2blfASO41375. (%) bb (A˚ ) Heme c binding site. Table 8 lists the top 25% of residues at the W(30) interface with 2blfBHEC1582 (heme c). The following table (Table K(7) 9) suggests possible disruptive replacements for these residues (see E(12) Section 4.6). V(3)T Table 8. N(1) res type subst’s cvg noc/ dist antn Q(2) (%) bb (A˚ ) A(3) 55 R V(2) 0.04 10/0 2.77 site G(1) R(92). D(2)M L(2)AGT IE Table 6. The top 25% of residues in 2blfA at the interface with sulfate 57 H H(72) 0.05 29/16 3.27 ion.(Field names: res: residue number in the PDB entry; type: amino acid N(15)T type; substs: substitutions seen in the alignment; with the percentage of each D(8).CR type in the bracket; noc/bb: number of contacts with the ligand, with the num- YF ber of contacts realized through backbone atoms given in the bracket; dist: 118 G .(12) 0.09 9/9 4.04 distance of closest apporach to the ligand. ) G(74) T(3) P(2) Table 7. Q(1) res type disruptive S(3)IEK mutations 56 Y S(4) 0.20 3/3 3.45 site 231 W (K)(E)(T)(R) N(44) W(7)D Table 7. List of disruptive mutations for the top 25% of residues in 2blfA, continued in next column that are at the interface with sulfate ion.

6 Table 8. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) T(1) H(15) Y(11) V(1) I(7). A(1)MCQ 231 W F(14) 0.23 3/0 4.44 Y(9) .(4) I(5) W(30) K(7) E(12) V(3)T N(1) Q(2) A(3) G(1) D(2)M Fig. 8. Residues in 2blfA, at the interface with heme c, colored by their rela- Table 8. The top 25% of residues in 2blfA at the interface with heme tive importance. The ligand (heme c) is colored green. Atoms further than A˚ c.(Field names: res: residue number in the PDB entry; type: amino acid type; 30 away from the geometric center of the ligand, as well as on the line of substs: substitutions seen in the alignment; with the percentage of each type sight to the ligand were removed. (See Appendix for the coloring scheme for in the bracket; noc/bb: number of contacts with the ligand, with the number of the protein chain 2blfA.) contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 9. res type disruptive mutations 55 R (Y)(D)(T)(E) 57 H (E)(M)(Q)(D) 118 G (R)(H)(FW)(K) 56 Y (K)(QR)(M)(E) 231 W (K)(E)(T)(R)

Fig. 9. A possible active surface on the chain 2blfA. The larger cluster it Table 9. List of disruptive mutations for the top 25% of residues in 2blfA, belongs to is shown in blue. that are at the interface with heme c.

Figure 8 shows residues in 2blfA colored by their importance, at the Table 10. continued interface with 2blfBHEC1582. res type substitutions(%) cvg 285 G G(99)Y 0.01 2.4.3 Possible novel functional surfaces at 25% coverage. One 47 T T(91)NI(2).M 0.07 group of residues is conserved on the 2blfA surface, away from (or V(1)KPG susbtantially larger than) other functional sites and interfaces reco- 343 N D(83).N(11)G(1) 0.07 gnizable in PDB entry 2blf. It is shown in Fig. 9. The right panel A(1)S(2) shows (in blue) the rest of the larger cluster this surface belongs to. 346 G G(74).M(4)A(4) 0.08 The residues belonging to this surface ”patch” are listed in Table 10, Y(8)K(2)L(1) while Table 11 suggests possible disruptive replacements for these F(2)SR residues (see Section 4.6). 190 G G(86)D(9)E(2)NS 0.12 Table 10. 288 F W(57)Y(9)F(20) 0.12 res type substitutions(%) cvg V(5)I(1)L(3)S 189 N N(99)E 0.01 M(1) continued in next column continued in next column

7 Table 10. continued The residues belonging to this surface ”patch” are listed in Table 12, res type substitutions(%) cvg while Table 13 suggests possible disruptive replacements for these 267 I V(10)TI(78)N 0.17 residues (see Section 4.6). L(3)F(2)M(1)A P(1). Table 12. 48 P P(85)S(3)A(2) 0.20 res type substitutions(%) cvg T(1).G(1)LEDNQV 307 W IW(98)FM 0.02 RF 304 G G(96)KSA(1)PE 0.04 321 F W(58)F(16)L(14) 0.20 340 R R(78)Q(1)K(19)V 0.07 H(1)AI(1)Y(2)D. L M(1)QG 302 D D(92)G(3)N(3)PE 0.08 289 D T(3)N(3)A(20) 0.22 300 S KS(79)T(5)C(2) 0.10 G(11)S(42)D(13) R(6)G(1)AQ(2)E CP(2).V 350 P P(84).(7)T(3) 0.11 286 I A(6)F(3)Y(26) 0.24 V(1)RSAQG V(15)I(17)H(5) L(11)W(4)R(3) Table 12. Residues forming surface ”patch” in 2blfA. M(1)ST(1)K(1)P

Table 13. Table 10. Residues forming surface ”patch” in 2blfA. res type disruptive mutations 307 W (KE)(T)(D)(QR) Table 11. 304 G (R)(K)(H)(FW) res type disruptive 340 R (TY)(D)(SECG)(FW) mutations 302 D (R)(H)(FW)(Y) 189 N (Y)(FWH)(T)(VCARG) 300 S (R)(FW)(H)(K) 285 G (K)(ER)(QM)(D) 350 P (Y)(R)(H)(E) 47 T (R)(H)(K)(FW) 343 N (Y)(H)(FW)(R) Table 13. Disruptive mutations for the surface patch in 2blfA. 346 G (E)(KR)(D)(H) 190 G (R)(K)(FWH)(YM) Another group of surface residues is shown in Fig.11. The right panel 288 F (K)(E)(QR)(TD) shows (in blue) the rest of the larger cluster this surface belongs to. 267 I (R)(Y)(H)(T) 48 P (Y)(R)(H)(T) 321 F (K)(E)(T)(Q) 289 D (R)(H)(FW)(K) 286 I (R)(Y)(T)(E)

Table 11. Disruptive mutations for the surface patch in 2blfA.

Another group of surface residues is shown in Fig.10. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 11. Another possible active surface on the chain 2blfA. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 14, while Table 15 suggests possible disruptive replacements for these residues (see Section 4.6). Table 14. res type substitutions(%) cvg antn 200 P P(100) 0.01 Fig. 10. Another possible active surface on the chain 2blfA. The larger cluster continued in next column it belongs to is shown in blue.

8 Table 14. continued Table 14. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn 202 R R(96)K(2)V 0.02 F(4)I(3)LG(6) 104 C C(96)V(2)QG.A 0.03 site V(13)SH(2)ENR 215 K K(94)T(3)C(1) 0.03 209 Y Y(31)I(13)F(7) 0.18 55 R V(2)R(92).L(2)A 0.04 site P(7)A(4)V(13) GTIE E(13)T(3)LS(2) 210 G G(86)A(11)S(2) 0.04 site Q(1)WD 57 H H(72)N(15)TD(8) 0.05 237 R V(2)R(34)H(2) 0.19 .CRYF F(2)W(7)K(21) 206 P P(89)G(7)F(1)QM 0.05 .(14)A(2)N(1) YL L(3)T(1)Q(3)IGS 236 Y Y(91)N(3)W(3).S 0.06 site 56 Y S(4)N(44)W(7)D 0.20 site 107 N N(90)E(1)LS(2)I 0.07 T(1)H(15)Y(11) T(3)D. V(1)I(7).A(1)MC 193 L L(86)MI(11)VA 0.08 Q 118 G .(12)G(74)T(3) 0.09 213 W W(34)S(38)N(15) 0.20 site P(2)Q(1)S(3)IEK Q(1)A(4)C(1) 197 N H(68)N(22)Q(7) 0.09 site H(2)M(1)F R(1)Y 54 V AV(48)I(9)RK(5) 0.21 109 R SR(70)V(7)G(11) 0.10 E(14)Y(9)CL(3) N(1)L(1).(1) T(2).D(1)GSNM C(1)APMTQ 194 P P(61)E(2)H(1) 0.22 356 N .(11)S(1)N(64) 0.11 T(5)N(4)LR(15) D(3)IR(9)A(1) S(5)KDYQA G(1)MV(1)HLPEF 214 V V(63)I(14)G(2)P 0.22 105 S S(31)A(43)G(12) 0.12 site C(2)T(2)Q(4) V(7)D(2)T(1)I. A(7)M(1)L 156 G G(75)A(3)DS(15) 0.12 231 W F(14)Y(9).(4) 0.23 N(2)FC.QE I(5)W(30)K(7) 53 F F(63)Y(34).LAV 0.13 site E(12)V(3)TN(1) 212 Y A(10)R(30)N(7) 0.13 Q(2)A(3)G(1) Y(16)V(13)L(3) D(2)M M(9)T(5)S(1)KCI 235 A D(28).(2)G(6) 0.23 211 T V(9)G(6)T(18) 0.14 site A(31)S(4)K(13) S(9)M(6)F(2) F(1)E(3)R(2) A(23)N(13)Y(7)D M(1)LP(1)TY I(2)E 120 Q P(13)G(7)S(3) 0.24 121 L TF(9)W(56).(10) 0.15 Q(46).(10)MK(1) D(1)L(11)Y(2)K A(2)T(1)E(3) V(1)NA(2)IQSE N(4)D(3)L 355 W .(11)W(56)E(3) 0.15 186 Y Y(49)W(22)H(1) 0.25 S(2)L(13)M(1) V(5)F(11)L(6) R(3)DTP(2)Y(2)G I(2)T F(1)H 208 Y W(33)V(13)Q(1) 0.25 158 D D(81)TK(1)E(7)A 0.16 Y(24)L(11)F(5)N G(3)CLQ(1).VH I(2)R(2)H(3) 70 F W(40)Y(29)H(17) 0.17 T(1)K F(9)L(2). 232 M M(27)H(4).(4) 0.17 Table 14. Residues forming surface ”patch” in 2blfA. Q(32)T(4)E(15) V(1)D(3)N(1)A R(1)I Table 15. 360 Y .(11)M(6)Y(47) 0.17 res type disruptive continued in next column mutations 200 P (YR)(TH)(SKECG)(FQWD) continued in next column

9 Table 15. continued res type disruptive mutations 202 R (TYD)(E)(SCG)(FVLAWPI) 104 C (E)(R)(K)(H) 215 K (FW)(Y)(H)(VA) 55 R (Y)(D)(T)(E) Fig. 12. Residues 501-581 in 2blfB colored by their relative importance. (See 210 G (KR)(E)(QH)(FMW) Appendix, Fig.18, for the coloring scheme.) 57 H (E)(M)(Q)(D) 206 P (R)(Y)(T)(H) 236 Y (K)(QM)(E)(R) to this report, under the name of 2blfB.msf. Its statistics, from the 107 N (Y)(H)(FW)(R) alistat program are the following: 193 L (Y)(R)(H)(T) 118 G (R)(H)(FW)(K) Format: MSF 197 N (TY)(FVAW)(E)(SCG) Number of sequences: 35 109 R (YD)(TE)(FW)(S) Total number of residues: 2727 356 N (Y)(H)(T)(R) Smallest: 70 105 S (R)(K)(H)(Q) Largest: 81 156 G (R)(K)(H)(E) Average length: 77.9 53 F (K)(E)(Q)(D) Alignment length: 81 212 Y (K)(R)(E)(Q) Average identity: 39% 211 T (R)(K)(H)(Q) Most related pair: 95% 121 L (R)(Y)(H)(T) Most unrelated pair: 12% 355 W (K)(E)(Q)(T) Most distant seq: 31% 158 D (R)(H)(FW)(Y) 70 F (K)(E)(Q)(T) Furthermore, 4% of residues show as conserved in this alignment. 232 M (Y)(H)(T)(R) The alignment consists of 11% prokaryotic sequences. (Descripti- 360 Y (K)(Q)(R)(M) ons of some sequences were not readily available.) The file contai- 209 Y (K)(R)(Q)(EM) ning the sequence descriptions can be found in the attachment, under 237 R (D)(T)(E)(Y) the name 2blfB.descr. 56 Y (K)(QR)(M)(E) 213 W (E)(K)(D)(T) 3.3 Residue ranking in 2blfB 54 V (R)(Y)(K)(E) The 2blfB sequence is shown in Fig. 12, with each residue colored 194 P (Y)(R)(H)(T) according to its estimated importance. The full listing of residues 214 V (R)(Y)(KE)(H) in 2blfB can be found in the file called 2blfB.ranks sorted in the 231 W (K)(E)(T)(R) attachment. 235 A (R)(Y)(K)(E) 120 Q (Y)(H)(FW)(T) 3.4 Top ranking residues in 2blfB and their position on 186 Y (K)(Q)(E)(R) the structure 208 Y (K)(E)(Q)(M) In the following we consider residues ranking among top 25% of residues in the protein . Figure 13 shows residues in 2blfB colored Table 15. Disruptive mutations for the surface patch in 2blfA. by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

3 CHAIN 2BLFB 3.4.1 Clustering of residues at 25% coverage. Fig. 14 shows the 3.1 Q9LA15 overview top 25% of all residues, this time colored according to clusters they From SwissProt, id Q9LA15, 100% identical to 2blfB: belong to. The clusters in Fig.14 are composed of the residues listed Description: Sulfite:cytochrome c oxidoreductase subunit B. in Table 16. Organism, scientific name: Thiobacillus novellus. Table 16. Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Rhizo- cluster size member biales; Hyphomicrobiaceae; Starkeya. color residues red 13 521,529,532,533,534,536,537 3.2 Multiple sequence alignment for 2blfB 541,542,550,576,577,581 For the chain 2blfB, the alignment 2blfB.msf (attached) with 35 blue 5 556,557,561,563,564 sequences was used. The alignment was downloaded from the HSSP continued in next column database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment

10 3.4.2 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 2blfA.Table 17 lists the top 25% of residues at the interface with 2blfA. The following table (Table 18) suggests possible disruptive replacements for these residues (see Section 4.6). Table 17. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 532 C C(100) 0.05 24/18 3.29 site 533 H H(100) 0.05 31/26 3.09 541 Q Q(88) 0.10 40/13 2.91 site N(8) I(2) 534 S S(85) 0.14 47/22 2.92 E(2) A(8) K(2) 542 P P(82) 0.15 50/11 3.58 S(8) G(5) Fig. 13. Residues in 2blfB, colored by their relative importance. Clockwise: A(2) front, back, top and bottom views. 564 P P(82) 0.16 1/0 4.81 Q(8) K(5) T(2) 537 Y M(11) 0.17 56/4 3.09 site Y(77) F(8) A(2) 508 P T(2) 0.18 21/2 3.35 P(82) G(2) D(2) E(2) V(2) .(2) 561 Y Y(62) 0.22 33/9 3.35 site F(31) L(2) .(2) 507 L F(5) 0.23 22/2 3.97 L(74) A(2) V(5) I(5) M(2) Fig. 14. Residues in 2blfB, colored according to the cluster they belong to: .(2) red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The 536 D G(14) 0.25 43/5 2.95 corresponding Pymol script is attached. D(62) Q(11) H(2) Table 16. continued continued in next column cluster size member color residues yellow 2 507,508

Table 16. Clusters of top ranking residues in 2blfB.

11 Table 17. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) S(2) .(2) T(2)

Table 17. The top 25% of residues in 2blfB at the interface with 2blfA. (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 of closest apporach to the ligand. )

Table 18. res type disruptive mutations 532 C (KER)(FQMWHD)(NYLPI)(SVA) 533 H (E)(TQMD)(SNKVCLAPIG)(YR) 541 Q (Y)(TH)(FW)(SCG) 534 S (R)(FWH)(KY)(QM) 542 P (R)(Y)(H)(K) Fig. 15. Residues in 2blfB, at the interface with 2blfA, colored by their rela- 564 P (Y)(HR)(T)(FW) tive importance. 2blfA is shown in backbone representation (See Appendix 537 Y (K)(Q)(ER)(NM) for the coloring scheme for the protein chain 2blfB.) 508 P (R)(Y)(H)(K) 561 Y (K)(Q)(EMR)(N) 507 L (YR)(TH)(K)(E) Table 19. continued 536 D (R)(FW)(H)(K) res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Table 18. List of disruptive mutations for the top 25% of residues in 556 K K(91) 0.11 9/0 2.90 site 2blfB, that are at the interface with 2blfA. T(5) R(2) 564 P P(82) 0.16 1/0 4.78 Figure 15 shows residues in 2blfB colored by their importance, at the Q(8) interface with 2blfA. K(5) Heme c binding site. Table 19 lists the top 25% of residues at the T(2) interface with 2blfBHEC1582 (heme c). The following table (Table 537 Y M(11) 0.17 56/11 2.74 site 20) suggests possible disruptive replacements for these residues (see Y(77) Section 4.6). F(8) Table 19. A(2) res type subst’s cvg noc/ dist antn 561 Y Y(62) 0.22 55/4 3.23 site (%) bb (A˚ ) F(31) 529 C C(100) 0.05 41/23 1.91 site L(2) 532 C C(100) 0.05 27/8 1.86 site .(2) 533 H H(100) 0.05 78/0 2.07 577 L L(100) 0.05 2/0 3.65 site Table 19. The top 25% of residues in 2blfB at the interface with heme 550 W W(97) 0.07 2/0 4.20 c.(Field names: res: residue number in the PDB entry; type: amino acid type; A(2) substs: substitutions seen in the alignment; with the percentage of each type 557 M M(97) 0.07 75/2 2.33 site in the bracket; noc/bb: number of contacts with the ligand, with the number of G(2) contacts realized through backbone atoms given in the bracket; dist: distance 563 A A(97) 0.09 12/5 3.67 of closest apporach to the ligand. ) C(2) 541 Q Q(88) 0.10 12/0 3.65 site N(8) I(2) continued in next column

12 Table 20. res type disruptive mutations 529 C (KER)(FQMWHD)(NYLPI)(SVA) 532 C (KER)(FQMWHD)(NYLPI)(SVA) 533 H (E)(TQMD)(SNKVCLAPIG)(YR) 577 L (YR)(TH)(SKECG)(FQWD) 550 W (KE)(QD)(TR)(N) 557 M (Y)(H)(R)(T) 563 A (KER)(Y)(QHD)(N) 541 Q (Y)(TH)(FW)(SCG) 556 K (Y)(FW)(T)(VA) 564 P (Y)(HR)(T)(FW) 537 Y (K)(Q)(ER)(NM) 561 Y (K)(Q)(EMR)(N)

Table 20. List of disruptive mutations for the top 25% of residues in 2blfB, that are at the interface with heme c.

Fig. 17. A possible active surface on the chain 2blfB.

Fig. 16. Residues in 2blfB, at the interface with heme c, colored by their relative importance. The ligand (heme c) is colored green. Atoms further than 30A˚ 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 2blfB.)

Figure 16 shows residues in 2blfB colored by their importance, at the interface with 2blfBHEC1582. 3.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2blfB surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2blf. It 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 4.6).

13 Table 21. in a chain], according to trace. The ET results are presented in the res type substitutions(%) cvg antn form of a table, usually limited to top 25% percent of residues (or 529 C C(100) 0.05 site to some nearby percentage), sorted by the strength of the presumed 532 C C(100) 0.05 site evolutionary pressure. (I.e., the smaller the coverage, the stronger the 533 H H(100) 0.05 pressure on the residue.) Starting from the top of that list, mutating a 577 L L(100) 0.05 site couple of residues should affect the protein somehow, with the exact 550 W W(97)A(2) 0.07 effects to be determined experimentally. 557 M M(97)G(2) 0.07 site 563 A A(97)C(2) 0.09 4.2 Known substitutions 541 Q Q(88)N(8)I(2) 0.10 site One of the table columns is “substitutions” - other amino acid types 556 K K(91)T(5)R(2) 0.11 site seen at the same position in the alignment. These amino acid types 534 S S(85)E(2)A(8) 0.14 may be interchangeable at that position in the protein, so if one wants K(2) to affect the protein by a point mutation, they should be avoided. For 542 P P(82)S(8)G(5) 0.15 example if the substitutions are “RVK” and the original protein has A(2) an R at that position, it is advisable to try anything, but RVK. Conver- 564 P P(82)Q(8)K(5) 0.16 sely, when looking for substitutions which will not affect the protein, T(2) one may try replacing, R with K, or (perhaps more surprisingly), with 537 Y M(11)Y(77)F(8) 0.17 site V. The percentage of times the substitution appears in the alignment A(2) is given in the immediately following bracket. No percentage is given 581 Y .(22)Y(74)H(2) 0.20 in the cases when it is smaller than 1%. This is meant to be a rough 561 Y Y(62)F(31)L(2) 0.22 site guide - due to rounding errors these percentages often do not add up .(2) to 100%. 536 D G(14)D(62)Q(11) 0.25 H(2)S(2).(2) 4.3 Surface T(2) To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 Table 21. Residues forming surface ”patch” in 2blfB. at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor Table 22. within 5A˚ from any of their heavy atoms. res type disruptive Note, however, that, if our picture of protein evolution is correct, mutations the neighboring residues which are not surface accessible might be 529 C (KER)(FQMWHD)(NYLPI)(SVA) equally important in maintaining the interaction specificity - they 532 C (KER)(FQMWHD)(NYLPI)(SVA) should not be automatically dropped from consideration when choo- 533 H (E)(TQMD)(SNKVCLAPIG)(YR) sing the set for mutagenesis. (Especially if they form a cluster with 577 L (YR)(TH)(SKECG)(FQWD) the surface residues.) 550 W (KE)(QD)(TR)(N) 4.4 Number of contacts 557 M (Y)(H)(R)(T) 563 A (KER)(Y)(QHD)(N) Another column worth noting is denoted “noc/bb”; it tells the num- 541 Q (Y)(TH)(FW)(SCG) ber of contacts heavy atoms of the residue in question make across 556 K (Y)(FW)(T)(VA) the interface, as well as how many of them are realized through the 534 S (R)(FWH)(KY)(QM) backbone atoms (if all or most contacts are through the backbone, 542 P (R)(Y)(H)(K) mutation presumably won’t have strong impact). Two heavy atoms ˚ 564 P (Y)(HR)(T)(FW) are considered to be “in contact” if their centers are closer than 5A. 537 Y (K)(Q)(ER)(NM) 4.5 Annotation 581 Y (K)(M)(Q)(EVLAPI) 561 Y (K)(Q)(EMR)(N) If the residue annotation is available (either from the pdb file or 536 D (R)(FW)(H)(K) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 22. Disruptive mutations for the surface patch in 2blfB. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 4.6 Mutation suggestions 4 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- 4.1 Coverage mentarity with the substitutions found in the alignment. Note that Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large

14 more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, COVERAGE DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond.

V The colors used to distinguish the residues by the estimated 100% 50% 30% 5% evolutionary pressure they experience can be seen in Fig. 18. 5.3 Credits 5.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number

V of residues, the average and range of the sequence lengths, and the RELATIVE IMPORTANCE alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of Fig. 18. Coloring scheme used to color residues by their relative importance. exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant tively [KHR], or negatively [DE] charged, aromatic [WFYH], seq” is calculated by finding the maximum pairwise identity (best long aliphatic chain [EKRQM], OH-group possession [SDETY ], relative) for all N sequences, then finding the minimum of these N and NH2 group possession [NQRK]. The suggestions are listed numbers (hence, the most outlying sequence). alistat is copyrighted according to how different they appear to be from the original amino by HHMI/Washington University School of Medicine, 1992-2001, acid, and they are grouped in round brackets if they appear equally and freely distributed under the GNU General Public License. disruptive. From left to right, each bracketed group of amino acid 5.3.2 CE To map ligand binding sites from different types resembles more strongly the original (i.e. is, presumably, less source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 5.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 5 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A , which is roughly the area needed for one water molecule to come in 5.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: by [email protected] November 18,2002,

• alignment# number of the position in the alignment http://www.cmbi.kun.nl/gv/dssp/descrip.html. • residue# residue number in the PDB file 5.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de • rank rank of the position according to older version of ET Daruvar, and C. Sander. ”The HSSP database of protein structure- • variability has two subfields: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1. number of different amino acids appearing in in this column of the alignment http://swift.cmbi.kun.nl/swift/hssp/

2. their type 5.3.5 LaTex The text for this report was processed using LATEX; • rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 5.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97.

5.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 5.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol

15 is an open-source application copyrighted by DeLano Scien- • 2blfA.complex.pdb - coordinates of 2blfA with all of its interac- tific LLC (2005). For more information about Pymol see ting partners http://pymol.sourceforge.net/. (Note for Windows • 2blfA.etvx - ET viewer input file for 2blfA users: the attached package needs to be unzipped for Pymol to read • 2blfA.cluster report.summary - Cluster report summary for the scripts and launch the viewer.) 2blfA 5.4 Note about ET Viewer • 2blfA.ranks - Ranks file in sequence order for 2blfA Dan Morgan from the Lichtarge lab has developed a visualization • 2blfA.clusters - Cluster descriptions for 2blfA tool specifically for viewing trace results. If you are interested, please • 2blfA.msf - the multiple sequence alignment used for the chain visit: 2blfA • 2blfA.descr - description of sequences used in 2blfA msf http://mammoth.bcm.tmc.edu/traceview/ • 2blfA.ranks sorted - full listing of residues and their ranking for The viewer is self-unpacking and self-installing. Input files to be used 2blfA with ETV (extension .etvx) can be found in the attachment to the • 2blfA.2blfB.if.pml - Pymol script for Figure 5 main report. • 2blfA.cbcvg - used by other 2blfA – related pymol scripts • 5.5 Citing this work 2blfA.2blfAMSS1374.if.pml - Pymol script for Figure 6 • The method used to rank residues and make predictions in this report 2blfA.2blfASO41375.if.pml - Pymol script for Figure 7 can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 2blfA.2blfBHEC1582.if.pml - Pymol script for Figure 8 Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 2blfB.complex.pdb - coordinates of 2blfB with all of its interac- by Importance” J. Mol. Bio. 336: 1265-82. For the original version ting partners of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 2blfB.etvx - ET viewer input file for 2blfB tionary Trace Method Defines Binding Surfaces Common to Protein • 2blfB.cluster report.summary - Cluster report summary for Families” J. Mol. Bio. 257: 342-358. 2blfB report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 2blfB.ranks - Ranks file in sequence order for 2blfB of service for comparative analysis of proteins.” Bioinformatics • 2blfB.clusters - Cluster descriptions for 2blfB 22:1656-7. • 2blfB.msf - the multiple sequence alignment used for the chain 2blfB 5.6 About report maker • 2blfB.descr - description of sequences used in 2blfB msf report maker was written in 2006 by Ivana Mihalek. The 1D ran- • king visualization program was written by Ivica Res.ˇ report maker 2blfB.ranks sorted - full listing of residues and their ranking for is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2blfB Houston. • 2blfB.2blfA.if.pml - Pymol script for Figure 15 • 2blfB.cbcvg - used by other 2blfB – related pymol scripts 5.7 Attachments • 2blfB.2blfBHEC1582.if.pml - Pymol script for Figure 16 The following files should accompany this report:

16