Pages 1–13 2e7z Evolutionary trace report by report maker February 13, 2010

4.3.1 Alistat 13 4.3.2 CE 13 4.3.3 DSSP 13 4.3.4 HSSP 13 4.3.5 LaTex 13 4.3.6 Muscle 13 4.3.7 Pymol 13 4.4 Note about ET Viewer 13 4.5 Citing this work 13 4.6 About report maker 13 4.7 Attachments 13

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2e7z): Title: Acetylene hydratase from pelobacter acetylenicus CONTENTS Compound: Mol id: 1; molecule: acetylene hydratase ahy; chain: a; ec: 4.2.1.71 1 Introduction 1 Organism, scientific name: Pelobacter Acetylenicus; 2e7z contains a single unique chain 2e7zA (727 residues long). 2 Chain 2e7zA 1 2.1 Q71EW5 overview 1 2.2 Multiple sequence alignment for 2e7zA 1 2.3 Residue ranking in 2e7zA 1 2.4 Top ranking residues in 2e7zA and their position on the structure 1 2 CHAIN 2E7ZA 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at 2.1 Q71EW5 overview 25% coverage. 3 From SwissProt, id Q71EW5, 98% identical to 2e7zA: 2.4.3 Possible novel functional surfaces at 25% Description: Acetylene hydratase Ahy. coverage. 7 Organism, scientific name: Pelobacter acetylenicus. Taxonomy: Bacteria; Proteobacteria; Deltaproteobacteria; Desulfu- 3 Notes on using trace results 11 romonadales; Pelobacteraceae; Pelobacter. 3.1 Coverage 11 3.2 Known substitutions 12 3.3 Surface 12 3.4 Number of contacts 12 3.5 Annotation 12 2.2 Multiple sequence alignment for 2e7zA 3.6 Mutation suggestions 12 For the chain 2e7zA, the alignment 2e7zA.msf (attached) with 44 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 12 database, and fragments shorter than 75% of the query as well as 4.1 File formats 12 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 12 to this report, under the name of 2e7zA.msf. Its statistics, from the 4.3 Credits 13 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 4-245 in 2e7zA colored by their relative importance. (See Fig. 3. Residues 488-730 in 2e7zA colored by their relative importance. (See Appendix, Fig.15, for the coloring scheme.) Appendix, Fig.15, for the coloring scheme.)

2.4 Top ranking residues in 2e7zA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 4 shows residues in 2e7zA colored 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.

Fig. 2. Residues 246-487 in 2e7zA colored by their relative importance. (See Appendix, Fig.15, for the coloring scheme.)

Format: MSF Number of sequences: 44 Total number of residues: 30237 Smallest: 648 Largest: 727 Average length: 687.2 Alignment length: 727 Average identity: 32% Most related pair: 98% Most unrelated pair: 21% Most distant seq: 34%

Furthermore, 1% of residues show as conserved in this alignment. The alignment consists of 2% archaean sequences. (Descriptions of some sequences were not readily available.) The file containing Fig. 4. Residues in 2e7zA, colored by their relative importance. Clockwise: front, back, top and bottom views. the sequence descriptions can be found in the attachment, under the name 2e7zA.descr.

2.4.1 Clustering of residues at 25% coverage. Fig. 5 shows the 2.3 Residue ranking in 2e7zA top 25% of all residues, this time colored according to clusters they The 2e7zA sequence is shown in Figs. 1–3, with each residue colored belong to. The clusters in Fig.5 are composed of the residues listed according to its estimated importance. The full listing of residues in Table 1. in 2e7zA can be found in the file called 2e7zA.ranks sorted in the attachment.

2 Table 1. continued cluster size member color residues 641,642,645,648,652,655,661 672 green 6 9,12,16,46,48,49

Table 1. Clusters of top ranking residues in 2e7zA.

2.4.2 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. MGD binding site. Table 2 lists the top 25% of residues at the interface with 2e7zAMGD801 (mgd). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 418 N N(95) 0.02 30/17 3.01 D(2) Fig. 5. Residues in 2e7zA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for E(2) the coloring scheme). Clockwise: front, back, top and bottom views. The 141 C C(88) 0.08 23/2 2.50 corresponding Pymol script is attached. S(4) I(4) T(2) Table 1. 604 G G(88) 0.08 1/1 4.67 cluster size member .(2) color residues T(4) red 79 141,154,170,171,197,199,200 P(2) 201,202,204,207,210,211,213 A(2) 214,217,218,219,220,221,223 445 F F(79) 0.11 3/1 4.31 224,225,226,227,231,232,233 W(9) 235,237,238,239,242,243,246 Y(9) 249,250,253,256,257,260,262 M(2) 265,268,269,270,276,279,280 443 D D(77) 0.12 48/13 2.65 281,283,284,288,289,294,298 E(22) 308,313,315,317,318,319,324 447 T T(77) 0.12 5/0 3.34 554,555,557,558,559,560,561 S(9) 562,563,566,573,575,576,577 N(13) 578,580 613 S S(79) 0.12 19/8 2.60 blue 71 61,62,65,66,68,70,73,74,78 A(4) 81,82,83,84,85,86,87,90,93 L(2) 94,102,104,111,114,124,125 Q(6) 129,130,131,132,133,406,407 T(6) 408,411,416,418,432,433,437 606 R R(72) 0.14 22/0 2.90 438,440,443,445,447,448,449 G(9) 453,454,456,457,458,464,465 S(2) 487,490,494,497,501,502,505 H(9) 606,613,616,622,628,676,677 K(4) 678,679,681,682 P(2) yellow 15 597,598,599,601,634,636,637 676 H H(65) 0.16 57/2 3.01 continued in next column continued in next column

3 Table 2. continued Table 3. res type subst’s cvg noc/ dist res type disruptive (%) bb (A˚ ) mutations R(11) 418 N (Y)(FWH)(T)(R) S(4) 141 C (R)(K)(E)(H) A(2) 604 G (R)(K)(E)(H) Y(11) 445 F (K)(E)(TQD)(R) V(2) 443 D (R)(FWH)(YVCAG)(K) G(2) 447 T (R)(K)(FWH)(M) 416 A G(43) 0.17 47/34 3.14 613 S (R)(K)(H)(FW) A(29) 606 R (D)(T)(YE)(SVCLAPIG) N(6) 676 H (E)(Q)(D)(M) S(18) 416 A (R)(K)(E)(Y) T(2) 111 T (KR)(Q)(H)(M) 111 T G(63) 0.22 12/1 2.64 465 R (TD)(Y)(EVA)(CLPIG) S(11) 114 N (Y)(FW)(H)(T) A(2) 424 A (R)(K)(Y)(EH) V(9) E(2) Table 3. List of disruptive mutations for the top 25% of residues in Y(2) 2e7zA, that are at the interface with MGD. H(2) T(4) L(2) 465 R E(2) 0.22 8/0 2.86 R(65) H(15) T(4) Q(4) K(2) Y(2) V(2) 114 N R(29) 0.24 39/13 2.93 N(11) K(11) .(18) M(2) S(6) G(13) H(2) T(4) 424 A S(9) 0.24 1/1 4.94 P(25) G(18) A(40) E(2) T(2) Fig. 6. Residues in 2e7zA, at the interface with MGD, colored by their rela- V(2) tive importance. The ligand (MGD) 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 Table 2. The top 25% of residues in 2e7zA at the interface with protein chain 2e7zA.) MGD.(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 num- Figure 6 shows residues in 2e7zA colored by their importance, at the ber of contacts realized through backbone atoms given in the bracket; dist: interface with 2e7zAMGD801. distance of closest apporach to the ligand. ) MGD binding site. Table 4 lists the top 25% of residues at the interface with 2e7zAMGD802 (mgd). The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6).

4 Table 4. Table 4. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) 200 D D(100) 0.01 164/31 1.80 S(2) 221 D D(100) 0.01 81/5 1.46 H(9) 219 G G(97) 0.02 29/29 2.88 K(4) R(2) P(2) 201 P P(97) 0.03 88/14 3.01 171 G G(84) 0.17 88/88 2.79 S(2) W(4) 12 C .(15) 0.06 5/0 4.65 S(2) C(81) A(6) H(2) N(2) 220 T T(86) 0.06 5/5 4.10 199 L V(52) 0.18 34/27 2.68 S(13) L(11) 48 K R(43) 0.07 23/0 2.94 I(36) K(50) 204 T N(9) 0.23 11/0 3.31 G(4) S(20) S(2) T(65) 218 Y S(6) 0.07 145/19 2.69 I(4) A(4) P(86) Table 4. The top 25% of residues in 2e7zA at the interface with Y(2) MGD.(Field names: res: residue number in the PDB entry; type: amino acid 141 C C(88) 0.08 12/0 3.05 type; substs: substitutions seen in the alignment; with the percentage of each S(4) type in the bracket; noc/bb: number of contacts with the ligand, with the num- I(4) ber of contacts realized through backbone atoms given in the bracket; dist: T(2) distance of closest apporach to the ligand. ) 604 G G(88) 0.08 6/6 3.56 .(2) T(4) Table 5. P(2) res type disruptive A(2) mutations 202 R R(86) 0.10 75/16 2.17 200 D (R)(FWH)(KYVCAG)(TQM) W(2) 221 D (R)(FWH)(KYVCAG)(TQM) K(2) 219 G (E)(D)(FKMW)(YQLPHIR) V(2) 201 P (R)(Y)(H)(K) S(2) 12 C (E)(K)(QMDR)(FW) Q(2) 220 T (KR)(FQMWH)(NELPI)(D) A(2) 48 K (Y)(FW)(T)(VAD) 170 I W(81) 0.11 43/23 3.18 218 Y (K)(QR)(EM)(N) F(9) 141 C (R)(K)(E)(H) L(2) 604 G (R)(K)(E)(H) I(4) 202 R (D)(TY)(E)(CG) D(2) 170 I (R)(Y)(T)(K) 294 A G(65) 0.14 3/2 4.43 294 A (R)(K)(E)(Y) T(6) 606 R (D)(T)(YE)(SVCLAPIG) S(11) 171 G (KE)(R)(QD)(H) N(2) 199 L (YR)(H)(T)(KE) W(6) 204 T (R)(K)(H)(FW) L(2) A(4) Table 5. List of disruptive mutations for the top 25% of residues in 606 R R(72) 0.14 175/31 2.56 2e7zA, that are at the interface with MGD. G(9) continued in next column Figure 7 shows residues in 2e7zA colored by their importance, at the interface with 2e7zAMGD802. MPD binding site. Table 6 lists the top 25% of residues at the interface with 2e7zMPD1004 (mpd). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

5 Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 16 C C(97) 0.02 5/5 3.95 .(2)

Table 6. The top 25% of residues in 2e7zA at the interface with MPD.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 7. res type disruptive mutations 16 C (KER)(FQMWHD)(NLPI)(Y)

Table 7. List of disruptive mutations for the top 25% of residues in 2e7zA, that are at the interface with MPD.

Fig. 7. Residues in 2e7zA, at the interface with MGD, colored by their rela- tive importance. The ligand (MGD) 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 2e7zA.)

Fig. 8. Residues in 2e7zA, at the interface with MPD, colored by their rela- tive importance. The ligand (MPD) 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 2e7zA.)

Figure 8 shows residues in 2e7zA colored by their importance, at the interface with 2e7zMPD1004. Tungsten ion binding site. Table 8 lists the top 25% of residues at the interface with 2e7zAW803 (tungsten ion). The following table (Table 9) suggests possible disruptive replacements for these residues (see Section 3.6).

6 Table 8. res type subst’s cvg noc/ dist (%) bb (A˚ ) 141 C C(88) 0.08 6/2 2.34 S(4) I(4) T(2) 606 R R(72) 0.14 1/0 4.87 G(9) S(2) H(9) K(4) P(2)

Table 8. The top 25% of residues in 2e7zA at the interface with tungs- ten ion.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 9. Fig. 9. Residues in 2e7zA, at the interface with tungsten ion, colored by their res type disruptive relative importance. The ligand (tungsten ion) is colored green. Atoms further mutations than 30A˚ away from the geometric center of the ligand, as well as on the line 141 C (R)(K)(E)(H) of sight to the ligand were removed. (See Appendix for the coloring scheme 606 R (D)(T)(YE)(SVCLAPIG) for the protein chain 2e7zA.)

Table 9. List of disruptive mutations for the top 25% of residues in 2e7zA, that are at the interface with tungsten ion. Table 10. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) Figure 9 shows residues in 2e7zA colored by their importance, at the N(6) interface with 2e7zAW803. V(4) Iron/sulfur cluster binding site. Table 10 lists the top 25% of F(2) residues at the interface with 2e7zASF4800 (iron/sulfur cluster). The W(6) following table (Table 11) suggests possible disruptive replacements S(2) for these residues (see Section 3.6).

Table 10. Table 10. The top 25% of residues in 2e7zA at the interface with res type subst’s cvg noc/ dist iron/sulfur cluster.(Field names: res: residue number in the PDB entry; type: (%) bb (A˚ ) amino acid type; substs: substitutions seen in the alignment; with the percen- 46 C C(100) 0.01 17/7 2.30 tage of each type in the bracket; noc/bb: number of contacts with the ligand, 16 C C(97) 0.02 16/5 2.30 with the number of contacts realized through backbone atoms given in the .(2) bracket; dist: distance of closest apporach to the ligand. ) 9 C .(29) 0.05 17/6 2.31 C(70) 12 C .(15) 0.06 21/11 2.28 Table 11. C(81) res type disruptive H(2) mutations 48 K R(43) 0.07 13/2 3.72 46 C (KER)(FQMWHD)(NYLPI)(SVA) K(50) 16 C (KER)(FQMWHD)(NLPI)(Y) G(4) 9 C (KER)(FQMWHD)(NLPI)(Y) S(2) 12 C (E)(K)(QMDR)(FW) 49 S C(25) 0.19 13/7 3.49 48 K (Y)(FW)(T)(VAD) P(2) 49 S (KR)(EQ)(H)(M) G(50) continued in next column Table 11. List of disruptive mutations for the top 25% of residues in 2e7zA, that are at the interface with iron/sulfur cluster.

7 Table 12. res type substitutions(%) cvg 102 G G(100) 0.01 130 G G(93)H(2)Q(4) 0.04 407 Y Y(88)P(4)R(2) 0.04 W(2)D(2) 406 P P(90)A(2)D(4) 0.06 E(2) 104 E E(72)R(2)Q(15) 0.18 S(4)N(4) 129 L L(25)Y(4)F(54) 0.18 I(15) 124 R S(4)R(70)A(4) 0.20 L(6)N(2)G(6) F(2)I(2) 125 F M(4)F(70)L(11) 0.21 T(4)Y(4)C(2) A(2) 505 M L(56)M(31)F(2) 0.21 C(2)G(2)V(2) I(2) 408 P P(65)Q(6)A(9) 0.23 Fig. 10. Residues in 2e7zA, at the interface with iron/sulfur cluster, colored K(11)R(6) by their relative importance. The ligand (iron/sulfur cluster) is colored green. 512 P E(4)P(59)L(9) 0.25 Atoms further than 30A˚ away from the geometric center of the ligand, as well F(2)W(9)N(2) as on the line of sight to the ligand were removed. (See Appendix for the T(2).(4)D(4) coloring scheme for the protein chain 2e7zA.) K(2)

Figure 10 shows residues in 2e7zA colored by their importance, at Table 12. Residues forming surface ”patch” in 2e7zA. the interface with 2e7zASF4800. 2.4.3 Possible novel functional surfaces at 25% coverage. One Table 13. group of residues is conserved on the 2e7zA surface, away from (or res type disruptive susbtantially larger than) other functional sites and interfaces reco- mutations gnizable in PDB entry 2e7z. It is shown in Fig. 11. The right panel 102 G (KER)(FQMWHD)(NYLPI)(SVA) shows (in blue) the rest of the larger cluster this surface belongs to. 130 G (E)(KD)(R)(FW) 407 Y (K)(Q)(M)(R) 406 P (R)(Y)(H)(T) 104 E (FW)(H)(Y)(VCAG) 129 L (R)(TKY)(E)(H) 124 R (D)(TYE)(SCG)(FVLAWPI) 125 F (K)(E)(QR)(D) 505 M (Y)(HR)(T)(SD) 408 P (Y)(T)(H)(R) 512 P (R)(Y)(TH)(CG)

Table 13. Disruptive mutations for the surface patch in 2e7zA.

Another group of surface residues is shown in Fig.12. The right panel Fig. 11. A possible active surface on the chain 2e7zA. The larger cluster it shows (in blue) the rest of the larger cluster this surface belongs to. 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 The residues belonging to this surface ”patch” are listed in Table 12, residues (see Section 3.6). while Table 13 suggests possible disruptive replacements for these Table 14. residues (see Section 3.6). res type substitutions(%) cvg 200 D D(100) 0.01 continued in next column

8 Table 14. continued res type substitutions(%) cvg L(2)W(2) 262 L V(11)L(13)P(75) 0.12 288 P P(81)S(9)G(2) 0.12 N(2)A(2)Q(2) 210 A A(86)C(6)S(6) 0.13 270 G G(59)W(36)S(2) 0.13 Q(2) 233 I I(86)F(2)L(6) 0.15 V(4) 235 E E(77)D(2)R(6) 0.15 Fig. 12. Another possible active surface on the chain 2e7zA. The larger N(9)Q(2)T(2) cluster it belongs to is shown in blue. 558 P P(75)V(13)A(4) 0.15 T(4)L(2) Table 14. continued 559 S T(43)S(54)D(2) 0.15 res type substitutions(%) cvg 217 R R(88)I(2)L(4) 0.16 221 D D(100) 0.01 N(4) 242 F F(95)Y(4) 0.01 308 R Q(9)R(59)H(25) 0.16 557 T T(100) 0.01 L(4)A(2) 578 P P(100) 0.01 171 G G(84)W(4)S(2) 0.17 219 G G(97)R(2) 0.02 A(6)N(2) 563 E E(95)Q(2)D(2) 0.02 231 V V(79)I(11)E(2) 0.17 201 P P(97)S(2) 0.03 A(4)L(2) 560 G G(90)R(6)K(2) 0.03 246 W W(65)Y(25)H(4) 0.17 577 L L(95)V(4) 0.04 C(2)F(2) 249 G G(93)A(4)V(2) 0.05 211 D D(65)E(18)T(4) 0.18 239 D D(93)N(2)P(2) 0.06 Q(4)G(2)A(2) A(2) K(2) 218 Y S(6)A(4)P(86) 0.07 268 I I(75)L(9)R(2) 0.18 Y(2) V(11)A(2) 284 A A(79)W(6)G(11) 0.07 207 A A(81)G(6)S(4) 0.19 V(2) T(4)K(2) 214 L L(79)I(9)C(6) 0.08 213 W H(29)W(54)V(9) 0.20 M(2)V(2) F(4)Y(2) 243 V V(86)L(2)I(9) 0.08 238 Y Y(70)A(9)Q(2) 0.20 C(2) F(6)M(2)T(4) 576 P P(81)G(4)A(13) 0.08 V(2)I(2) 237 L L(81)W(13)K(2) 0.09 289 A A(63)G(11)L(6) 0.20 I(2) C(4)T(2)S(11) 317 V T(86)I(2)V(4) 0.09 256 R R(47)Y(11)H(25) 0.21 L(2)S(2)C(2) L(4)Q(4)M(2) 202 R R(86)W(2)K(2) 0.10 V(2)A(2) V(2)S(2)Q(2) 580 Y Y(68)W(2)H(13) 0.21 A(2) P(4).(2)F(9) 260 Y M(4)Y(65)F(27) 0.10 204 T N(9)S(20)T(65) 0.23 W(2) I(4) 561 K K(79)L(6)R(4) 0.10 281 V R(79)I(2)H(4) 0.24 M(9) E(4)Q(2)T(2) 573 G G(88)N(4)D(4) 0.10 Y(2)V(2) E(2) 575 D D(59)E(11)P(15) 0.24 170 I W(81)F(9)L(2) 0.11 S(9)A(2)N(2) I(4)D(2) 569 F F(27)M(6)L(54) 0.25 250 F F(90)G(2)Y(2) 0.11 V(2)A(4)Y(4) continued in next column continued in next column

9 Table 14. continued Table 15. continued res type substitutions(%) cvg res type disruptive mutations Table 14. Residues forming surface ”patch” in 2e7zA. 256 R (TD)(YE)(SCG)(VA) 580 Y (K)(Q)(E)(M) 204 T (R)(K)(H)(FW) Table 15. 281 V (KYER)(HD)(Q)(N) res type disruptive 575 D (R)(H)(FW)(Y) mutations 569 F (K)(E)(D)(QR) 200 D (R)(FWH)(KYVCAG)(TQM) 221 D (R)(FWH)(KYVCAG)(TQM) Table 15. Disruptive mutations for the surface patch in 2e7zA. 242 F (K)(E)(Q)(D) 557 T (KR)(FQMWH)(NELPI)(D) Another group of surface residues is shown in Fig.13. The right panel 578 P (YR)(TH)(SKECG)(FQWD) shows (in blue) the rest of the larger cluster this surface belongs to. 219 G (E)(D)(FKMW)(YQLPHIR) 563 E (FWH)(Y)(VCAG)(R) 201 P (R)(Y)(H)(K) 560 G (E)(D)(FW)(Y) 577 L (YR)(H)(TKE)(SQCDG) 249 G (KER)(QHD)(Y)(FMW) 239 D (R)(H)(Y)(FW) 218 Y (K)(QR)(EM)(N) 284 A (KE)(R)(Y)(QD) 214 L (R)(Y)(H)(T) 243 V (R)(Y)(KE)(H) 576 P (R)(Y)(H)(KE) 237 L (Y)(TR)(H)(SECG) 317 V (R)(K)(Y)(E) 202 R (D)(TY)(E)(CG) Fig. 13. Another possible active surface on the chain 2e7zA. The larger 260 Y (K)(Q)(E)(R) cluster it belongs to is shown in blue. 561 K (Y)(T)(FW)(SCG) 573 G (R)(FWH)(K)(Y) The residues belonging to this surface ”patch” are listed in Table 16, 170 I (R)(Y)(T)(K) while Table 17 suggests possible disruptive replacements for these 250 F (K)(E)(Q)(D) residues (see Section 3.6). 262 L (YR)(H)(T)(KE) 288 P (Y)(R)(H)(TE) Table 16. 210 A (KR)(E)(Y)(QH) res type substitutions(%) cvg 270 G (KER)(HD)(FQMW)(Y) 598 P P(97)S(2) 0.02 233 I (R)(Y)(T)(KEH) 655 G G(93)A(4)S(2) 0.04 235 E (FW)(H)(Y)(VA) 597 Y Y(84)F(15) 0.10 558 P (R)(Y)(H)(K) 634 P P(86)T(2)K(4) 0.13 559 S (R)(K)(FWH)(QM) R(4)L(2) 217 R (T)(Y)(D)(SECG) 636 T T(61)D(25)A(6) 0.13 308 R (T)(D)(Y)(E) V(4)K(2) 171 G (KE)(R)(QD)(H) 599 L F(11)L(77)Y(9) 0.15 231 V (YR)(K)(H)(E) M(2) 246 W (KE)(Q)(D)(T) 652 T N(31)T(50)S(15) 0.22 211 D (R)(FW)(H)(Y) R(2) 268 I (Y)(R)(T)(H) 207 A (R)(K)(E)(Y) Table 16. Residues forming surface ”patch” in 2e7zA. 213 W (E)(K)(Q)(D) 238 Y (K)(R)(Q)(E) 289 A (R)(K)(E)(Y) Table 17. continued in next column res type disruptive mutations 598 P (R)(Y)(H)(K) 655 G (KR)(E)(QH)(FMW) continued in next column

10 Table 17. continued Table 18. continued res type disruptive res type substitutions(%) cvg mutations 66 L M(31)L(65)A(2) 0.11 597 Y (K)(Q)(EM)(NR) 445 F F(79)W(9)Y(9) 0.11 634 P (Y)(R)(T)(H) M(2) 636 T (R)(K)(H)(FW) 679 W W(84)S(6)Y(2) 0.11 599 L (R)(TY)(K)(EH) T(4)A(2) 652 T (FKWR)(MH)(EQ)(LPI) 443 D D(77)E(22) 0.12 447 T T(77)S(9)N(13) 0.12 Table 17. Disruptive mutations for the surface patch in 2e7zA. 613 S S(79)A(4)L(2) 0.12 Q(6)T(6) 490 E E(79)D(13)Q(4) 0.14 Another group of surface residues is shown in Fig.14. The right panel V(2) shows (in blue) the rest of the larger cluster this surface belongs to. 606 R R(72)G(9)S(2) 0.14 H(9)K(4)P(2) 437 D T(2)D(68)E(27) 0.15 P(2) 676 H H(65)R(11)S(4) 0.16 A(2)Y(11)V(2) G(2) 416 A G(43)A(29)N(6) 0.17 S(18)T(2) 678 R W(81)S(2)Y(4) 0.17 N(2)H(2)G(2) F(2)R(2) 682 E E(72)D(2)L(2) 0.18 C(6)N(6)G(2) Fig. 14. Another possible active surface on the chain 2e7zA. The larger Y(2)V(2)A(2) cluster it belongs to is shown in blue. 93 K N(9)K(68)C(2) 0.19 R(18)E(2) The residues belonging to this surface ”patch” are listed in Table 18, 681 P P(72)G(6)E(6) 0.19 while Table 19 suggests possible disruptive replacements for these A(9)S(4) residues (see Section 3.6). 62 V I(38)L(43)V(18) 0.20 82 S T(22)S(65)G(6) 0.21 Table 18. P(4) res type substitutions(%) cvg 73 R R(68)K(18)G(4) 0.22 61 R R(100) 0.01 D(2)Q(2).(4) 65 P P(100) 0.01 111 T G(63)S(11)A(2) 0.22 418 N N(95)D(2)E(2) 0.02 V(9)E(2)Y(2) 83 W W(95)Y(4) 0.03 H(2)T(4)L(2) 454 D E(2)D(95)H(2) 0.03 465 R E(2)R(65)H(15) 0.22 68 N R(90)K(6)N(2) 0.04 T(4)Q(4)K(2) 78 W W(79)F(20) 0.04 Y(2)V(2) 85 Q E(59)Q(36)D(2) 0.06 497 F A(2)F(36)I(34) 0.22 R(2) L(13)V(13) 622 R R(84)A(4)Q(4) 0.07 84 D D(65)E(29)A(2) 0.23 K(4)L(2) K(2) 628 P P(93)T(2)Q(2) 0.07 114 N R(29)N(11)K(11) 0.24 A(2) .(18)M(2)S(6) 70 G R(9)G(77)P(9) 0.09 G(13)H(2)T(4) D(2)A(2) 67 K K(72)R(15)L(6) 0.25 453 A C(2)A(84)S(11) 0.09 I(2)M(2) G(2) 81 I C(2)I(88)A(2) 0.10 Table 18. Residues forming surface ”patch” in 2e7zA. V(4)T(2) continued in next column

11 Table 19. 3.2 Known substitutions res type disruptive One of the table columns is “substitutions” - other amino acid types mutations seen at the same position in the alignment. These amino acid types 61 R (TD)(SYEVCLAPIG)(FMW)(N) may be interchangeable at that position in the protein, so if one wants 65 P (YR)(TH)(SKECG)(FQWD) to affect the protein by a point mutation, they should be avoided. For 418 N (Y)(FWH)(T)(R) example if the substitutions are “RVK” and the original protein has 83 W (K)(E)(Q)(D) an R at that position, it is advisable to try anything, but RVK. Conver- 454 D (R)(FW)(VCAHG)(K) sely, when looking for substitutions which will not affect the protein, 68 N (Y)(T)(FW)(SVCAG) one may try replacing, R with K, or (perhaps more surprisingly), with 78 W (KE)(TQD)(SNCRG)(M) V. The percentage of times the substitution appears in the alignment 85 Q (Y)(FW)(TH)(VCAG) is given in the immediately following bracket. No percentage is given 622 R (TY)(D)(SECG)(FW) in the cases when it is smaller than 1%. This is meant to be a rough 628 P (YR)(H)(TKE)(FW) guide - due to rounding errors these percentages often do not add up 70 G (R)(KE)(H)(Y) to 100%. 453 A (KR)(E)(Y)(QH) 81 I (R)(Y)(H)(K) 3.3 Surface 66 L (Y)(R)(H)(T) To detect candidates for novel functional interfaces, first we look for 445 F (K)(E)(TQD)(R) residues that are solvent accessible (according to DSSP program) by 2 679 W (K)(E)(Q)(R) at least 10A˚ , which is roughly the area needed for one water mole- 443 D (R)(FWH)(YVCAG)(K) cule to come in the contact with the residue. Furthermore, we require 447 T (R)(K)(FWH)(M) that these residues form a “cluster” of residues which have neighbor 613 S (R)(K)(H)(FW) within 5A˚ from any of their heavy atoms. 490 E (H)(FW)(Y)(R) Note, however, that, if our picture of protein evolution is correct, 606 R (D)(T)(YE)(SVCLAPIG) the neighboring residues which are not surface accessible might be 437 D (R)(H)(FW)(KY) equally important in maintaining the interaction specificity - they 676 H (E)(Q)(D)(M) should not be automatically dropped from consideration when choo- 416 A (R)(K)(E)(Y) sing the set for mutagenesis. (Especially if they form a cluster with 678 R (D)(E)(T)(LPI) the surface residues.) 682 E (H)(R)(FW)(Y) 93 K (Y)(FW)(T)(VA) 3.4 Number of contacts 681 P (R)(Y)(H)(K) Another column worth noting is denoted “noc/bb”; it tells the num- 62 V (YR)(KE)(H)(QD) ber of contacts heavy atoms of the residue in question make across 82 S (R)(K)(H)(FQW) the interface, as well as how many of them are realized through the 73 R (T)(Y)(D)(VA) backbone atoms (if all or most contacts are through the backbone, 111 T (KR)(Q)(H)(M) mutation presumably won’t have strong impact). Two heavy atoms 465 R (TD)(Y)(EVA)(CLPIG) are considered to be “in contact” if their centers are closer than 5A˚ . 497 F (KE)(R)(Q)(TD) 84 D (R)(FWH)(Y)(CG) 3.5 Annotation 114 N (Y)(FW)(H)(T) If the residue annotation is available (either from the pdb file or 67 K (Y)(T)(FW)(SCG) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 19. Disruptive mutations for the surface patch in 2e7zA. (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 3.1 Coverage they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less

12 4.3 Credits 4.3.1 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 COVERAGE of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are V some percent identities. A percent pairwise alignment identity is defi- 100% 50% 30% 5% ned as (idents / MIN(len1, len2)) where idents is the number of 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, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best V relative) for all N sequences, then finding the minimum of these N RELATIVE IMPORTANCE numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License. Fig. 15. Coloring scheme used to color residues by their relative importance. 4.3.2 CE To map ligand binding sites from different 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. 4.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 4 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 4.1 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 • type amino acid type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than • rank rank of the position according to older version of ET 75% of the query are taken out, however); R. Schneider, A. de • variability has two subfields: Daruvar, and C. Sander. ”The HSSP database of protein structure- 1. number of different amino acids appearing in in this column sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of the alignment 2. their type http://swift.cmbi.kun.nl/swift/hssp/ • rho ET score - the smaller this value, the lesser variability of this position across the branches of the tree (and, presumably, 4.3.5 LaTex The text for this report was processed using LATEX; the greater the importance for the protein) Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 4.3.6 Muscle When making alignments “from scratch”, report • gaps percentage of gaps in this column maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and 4.2 Color schemes used high throughput.” Nucleic Acids Research 32(5), 1792-97. The following color scheme is used in figures with residues colored by cluster size: black is a single-residue cluster; clusters composed of http://www.drive5.com/muscle/ more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.7 Pymol The figures in this report were produced using quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, Pymol. The scripts can be found in the attachment. Pymol bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, is an open-source application copyrighted by DeLano Scien- DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tific LLC (2005). For more information about Pymol see tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. http://pymol.sourceforge.net/. (Note for Windows The colors used to distinguish the residues by the estimated users: the attached package needs to be unzipped for Pymol to read evolutionary pressure they experience can be seen in Fig. 15. the scripts and launch the viewer.)

13 4.4 Note about ET Viewer 4.7 Attachments Dan Morgan from the Lichtarge lab has developed a visualization The following files should accompany this report: tool specifically for viewing trace results. If you are interested, please • 2e7zA.complex.pdb - coordinates of 2e7zA with all of its visit: interacting partners http://mammoth.bcm.tmc.edu/traceview/ • 2e7zA.etvx - ET viewer input file for 2e7zA The viewer is self-unpacking and self-installing. Input files to be used • 2e7zA.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 2e7zA main report. • 2e7zA.ranks - Ranks file in sequence order for 2e7zA 4.5 Citing this work • 2e7zA.clusters - Cluster descriptions for 2e7zA The method used to rank residues and make predictions in this report • 2e7zA.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 2e7zA Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 2e7zA.descr - description of sequences used in 2e7zA msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 2e7zA.ranks sorted - full listing of residues and their ranking for of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 2e7zA tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 2e7zA.2e7zAMGD801.if.pml - Pymol script for Figure 6 report maker itself is described in Mihalek I., I. Res and O. • 2e7zA.cbcvg - used by other 2e7zA – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 2e7zA.2e7zAMGD802.if.pml - Pymol script for Figure 7 of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 2e7zA.2e7zMPD1004.if.pml - Pymol script for Figure 8 • 2e7zA.2e7zAW803.if.pml - Pymol script for Figure 9 4.6 About report maker • 2e7zA.2e7zASF4800.if.pml - Pymol script for Figure 10 report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston.

14