Pages 1–11 3na0 Evolutionary trace report by report maker September 13, 2010

4.3.1 Alistat 10 4.3.2 CE 10 4.3.3 DSSP 10 4.3.4 HSSP 11 4.3.5 LaTex 11 4.3.6 Muscle 11 4.3.7 Pymol 11 4.4 Note about ET Viewer 11 4.5 Citing this work 11 4.6 About report maker 11 4.7 Attachments 11

1 INTRODUCTION From the original Data Bank entry (PDB id 3na0): Title: Crystal structure of human cyp11a1 in complex with 20,22- dihydroxycholesterol Compound: Mol id: 1; molecule: cholesterol side-chain cleavage enzyme, mitochond chain: a, b; fragment: unp residues 44-514; syn- onym: 11a1, cypxia1, cytochrome p450(scc chole- sterol desmolase; ec: 1.14.15.6; engineered: yes; mol id: 2; molecule: CONTENTS adrenodoxin, mitochondrial; chain: c, d; fragment: unp residues 88-155; synonym: adrenal , ferredoxin-1, hepatoredoxin; 1 Introduction 1 engineered: yes Organism, scientific name: Homo Sapiens; 2 Chain 3na0A 1 3na0 contains a single unique chain 3na0A (470 residues long) and 2.1 Q8N1A7 overview 1 its homologue 3na0B. Not enough homologous sequences could be 2.2 Multiple sequence alignment for 3na0A 1 found to permit analysis for chain 3na0C. 2.3 Residue ranking in 3na0A 1 2.4 Top ranking residues in 3na0A and their position on the structure 2 2 CHAIN 3NA0A 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at 2.1 Q8N1A7 overview 25% coverage. 2 From SwissProt, id Q8N1A7, 100% identical to 3na0A: 2.4.3 Possible novel functional surfaces at 25% Description: Cytochrome P450, subfamily XIA,. coverage. 6 Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3 Notes on using trace results 9 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 3.1 Coverage 9 Catarrhini; Hominidae; Homo. 3.2 Known substitutions 9 Similarity: Belongs to the cytochrome P450 family. 3.3 Surface 9 3.4 Number of contacts 10 3.5 Annotation 10 2.2 Multiple sequence alignment for 3na0A 3.6 Mutation suggestions 10 For the chain 3na0A, the alignment 3na0A.msf (attached) with 118 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 10 database, and fragments shorter than 75% of the query as well as 4.1 File formats 10 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 10 to this report, under the name of 3na0A.msf. Its statistics, from the 4.3 Credits 10 alistat program are the following:

1 Lichtarge lab 2006 2.4 Top ranking residues in 3na0A and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 3na0A 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. 1. Residues 45-279 in 3na0A colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.)

Fig. 2. Residues 280-514 in 3na0A colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.) Fig. 3. Residues in 3na0A, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 118 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 53345 top 25% of all residues, this time colored according to clusters they Smallest: 356 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 470 in Table 1. Average length: 452.1 Alignment length: 470 Table 1. Average identity: 37% cluster size member Most related pair: 99% color residues Most unrelated pair: 17% red 113 85,86,87,88,89,99,114,115 Most distant seq: 32% 118,120,126,129,130,138,139 144,147,151,154,155,159,167 174,177,178,203,205,208,213 Furthermore, 1% of residues show as conserved in this alignment. 215,216,217,218,220,221,222 The alignment consists of 48% eukaryotic ( 47% vertebrata) 233,234,263,266,270,271,273 sequences. (Descriptions of some sequences were not readily availa- 302,305,311,315,318,319,322 ble.) The file containing the sequence descriptions can be found in 323,326,327,328,329,330,331 the attachment, under the name 3na0A.descr. 334,335,337,339,340,342,343 344,346,349,352,355,356,359 2.3 Residue ranking in 3na0A 375,376,377,378,379,382,383 384,385,386,387,389,390,396 The 3na0A 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 3na0A can be found in the file called 3na0A.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 466 R R(94) 0.09 16/0 3.37 .(4)F 465 R R(87) 0.12 9/0 3.40 K(7)T .(4) 159 M L(54) 0.13 22/9 3.34 M(35) F(2) I(6)H 378 K R(32) 0.13 15/0 3.27 K(66)W 450 F F(87) 0.14 46/7 3.36 Y(6)T S(1)R .(1)A 155 N N(71) 0.16 26/8 3.64 S(4) A(5) R(1) Fig. 4. Residues in 3na0A, colored according to the cluster they belong to: Q(10) red, followed by blue and yellow are the largest clusters (see Appendix for D(5)G the coloring scheme). Clockwise: front, back, top and bottom views. The 461 Q Q(54) 0.20 18/0 2.97 corresponding Pymol script is attached. S(10) A(9) M(19) Table 1. continued G(1)R cluster size member .(4) color residues 469 E E(88) 0.23 4/0 4.31 401,404,407,409,410,411,413 Q(3) 415,423,429,432,435,437,439 .(5)GV 440,454,455,456,458,460,461 462,464,465,466,468,469,471 blue 2 104,450 Table 2. The top 25% of residues in 3na0A at the interface with 3na0C. (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 Table 1. Clusters of top ranking residues in 3na0A. 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. ) 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. Table 3. Interface with 3na0C.Table 2 lists the top 25% of residues at the res type disruptive interface with 3na0C. The following table (Table 3) suggests possible mutations disruptive replacements for these residues (see Section 3.6). 151 R (TD)(SYEVCLAPIG)(FMW)(N) 382 K (Y)(T)(FW)(SVCAG) Table 2. 458 G (KER)(FQMWHD)(NLPI)(Y) res type subst’s cvg noc/ dist antn 456 G (K)(ER)(Q)(MD) A˚ (%) bb ( ) 466 R (TD)(SECG)(VLAPI)(Y) 151 R R(100) 0.01 1/0 4.84 465 R (D)(T)(YVLAPI)(SFECWG) 382 K K(96) 0.03 9/0 2.66 159 M (Y)(T)(R)(S) R(3) 378 K (TY)(SCG)(FVAWD)(E) 458 G G(96) 0.05 13/13 3.12 450 F (K)(E)(Q)(D) .(3) 155 N (Y)(FWH)(T)(R) 456 G G(94)W 0.07 1/1 4.85 site 461 Q (Y)(H)(FW)(T) .(3)S 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˚ ) 469 E (H)(FW)(Y)(R) 460 R R(94) 0.06 38/17 2.73 site .(4)P Table 3. List of disruptive mutations for the top 25% of residues in 462 C C(94)S 0.06 70/33 2.33 site 3na0A, that are at the interface with 3na0C. .(4) 464 G G(94)R 0.06 35/35 3.53 site .(4) 456 G G(94)W 0.07 10/10 3.46 site .(3)S 385 L L(85) 0.08 5/0 3.60 site M(8) F(2)IQE S 468 A A(94) 0.09 10/2 3.76 .(4)P 331 T T(83) 0.10 9/4 4.08 V(9) A(3)I S(2) 323 M L(74) 0.12 13/7 3.25 site M(19) V(3) I(1)G 334 T P(22) 0.12 5/0 3.37 site T(59)V S(13)G A(1)D 327 G S(19) 0.13 12/12 3.57 site G(66) A(14) Fig. 5. Residues in 3na0A, at the interface with 3na0C, colored by their rela- 322 E D(13) 0.17 3/1 4.47 tive importance. 3na0C is shown in backbone representation (See Appendix E(83)GP for the coloring scheme for the protein chain 3na0A.) K 326 G G(69) 0.18 23/23 3.68 site Figure 5 shows residues in 3na0A colored by their importance, at the A(28).H interface with 3na0C. 390 I V(65) 0.20 17/2 3.65 site HEM binding site. Table 4 lists the top 25% of residues at the I(22) interface with 3na0AHEM601 (hem). The following table (Table S(8) 5) suggests possible disruptive replacements for these residues (see G(1) Section 3.6). T(1)A 461 Q Q(54) 0.20 20/18 3.69 Table 4. S(10) res type subst’s cvg noc/ dist antn A(9) (%) bb (A˚ ) M(19) 151 R R(100) 0.01 12/0 2.67 G(1)R 396 R R(100) 0.01 10/0 2.90 site .(4) 147 W W(97) 0.02 12/0 2.85 site 139 V V(67) 0.24 16/6 3.90 .(1)V L(20) 120 R R(97) 0.04 45/0 2.67 site I(1) .(1)E P(6) 455 F F(94)WL 0.05 50/24 3.51 .(2)E .(3) 454 G A(22) 0.24 12/12 3.21 site 330 T T(94) 0.06 37/8 3.29 P(42) S(4)A continued in next column continued in next column

4 Table 4. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) G(27)V S(1) T(2) .(3)

Table 4. The top 25% of residues in 3na0A 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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 5. res type disruptive mutations 151 R (TD)(SYEVCLAPIG)(FMW)(N) 396 R (TD)(SYEVCLAPIG)(FMW)(N) 147 W (E)(K)(D)(Q) 120 R (T)(VCAG)(Y)(SLPDI) Fig. 6. Residues in 3na0A, at the interface with HEM, colored by their rela- 455 F (KE)(T)(QD)(SCRG) tive importance. The ligand (HEM) is colored green. Atoms further than 30A˚ 330 T (KR)(QH)(FMW)(E) away from the geometric center of the ligand, as well as on the line of sight 460 R (T)(D)(CG)(Y) to the ligand were removed. (See Appendix for the coloring scheme for the 462 C (KR)(E)(FMWH)(Q) protein chain 3na0A.) 464 G (E)(D)(KM)(FW) 456 G (K)(ER)(Q)(MD) 385 L (Y)(R)(H)(T) 468 A (Y)(R)(KE)(H) 331 T (R)(K)(H)(Q) Table 6. continued 323 M (Y)(H)(R)(T) res type subst’s cvg noc/ dist antn 334 T (R)(K)(H)(Q) (%) bb (A˚ ) 327 G (KR)(E)(QH)(FMW) 330 T T(94) 0.06 4/0 3.97 322 E (FWH)(Y)(R)(VCAG) S(4)A 326 G (E)(K)(R)(QD) 126 W W(83) 0.14 15/0 4.17 390 I (R)(Y)(H)(K) M(3) 461 Q (Y)(H)(FW)(T) .(2)D 139 V (YR)(KH)(E)(Q) I(2) 454 G (R)(K)(E)(H) L(5) F(2) Table 5. List of disruptive mutations for the top 25% of residues in 270 W W(88)Q 0.15 4/0 4.45 3na0A, that are at the interface with HEM. S(4)GFR M(1)LIH Figure 6 shows residues in 3na0A colored by their importance, at the 322 E D(13) 0.17 10/6 3.73 interface with 3na0AHEM601. E(83)GP 2DC binding site. Table 6 lists the top 25% of residues at the K interface with 3na0A2DC602 (2dc). The following table (Table 7) 326 G G(69) 0.18 16/16 3.57 site suggests possible disruptive replacements for these residues (see A(28).H Section 3.6). 390 I V(65) 0.20 4/1 4.46 site I(22) Table 6. S(8) res type subst’s cvg noc/ dist antn G(1) (%) bb (A˚ ) T(1)A 120 R R(97) 0.04 9/0 3.92 site .(1)E Table 6. continued in next column The top 25% of residues in 3na0A at the interface with 2DC.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each

5 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 120 R (T)(VCAG)(Y)(SLPDI) 330 T (KR)(QH)(FMW)(E) 126 W (K)(T)(E)(R) 270 W (E)(K)(T)(D) Fig. 8. A possible active surface on the chain 3na0A. The larger cluster it 322 E (FWH)(Y)(R)(VCAG) belongs to is shown in blue. 326 G (E)(K)(R)(QD) 390 I (R)(Y)(H)(K) 8, while Table 9 suggests possible disruptive replacements for these Table 7. List of disruptive mutations for the top 25% of residues in residues (see Section 3.6). 3na0A, that are at the interface with 2DC. Table 8. res type substitutions(%) cvg 407 Y Y(97)F(1)H 0.03 86 G G(94).(4)D 0.06 88 I I(81)M(5)V(5) 0.19 L(4).(4) 87 P P(79)R.(4)K(9)D 0.20 NS(2)Q(1) 85 Y L(27)Y(45).(4) 0.25 F(21)H

Table 8. Residues forming surface ”patch” in 3na0A.

Table 9. res type disruptive mutations 407 Y (K)(Q)(EM)(N) 86 G (R)(K)(FWH)(M) 88 I (Y)(R)(H)(T) 87 P (Y)(THR)(CG)(SFW) 85 Y (K)(Q)(EM)(NR)

Table 9. Disruptive mutations for the surface patch in 3na0A.

Fig. 7. Residues in 3na0A, at the interface with 2DC, colored by their relative Another group of surface residues is shown in Fig.9. The right panel importance. The ligand (2DC) is colored green. Atoms further than 30A˚ away shows (in blue) the rest of the larger cluster this surface belongs to. from the geometric center of the ligand, as well as on the line of sight to the The residues belonging to this surface ”patch” are listed in Table 10, ligand were removed. (See Appendix for the coloring scheme for the protein while Table 11 suggests possible disruptive replacements for these chain 3na0A.) residues (see Section 3.6). Table 10. Figure 7 shows residues in 3na0A colored by their importance, at the res type substitutions(%) cvg interface with 3na0A2DC602. 218 R R(95).H(1)KQ 0.04 220 G G(95).PD(2) 0.06 2.4.3 Possible novel functional surfaces at 25% coverage. One 177 D D(91)E(3)N(1)K 0.11 group of residues is conserved on the 3na0A surface, away from (or S(1)A susbtantially larger than) other functional sites and interfaces reco- 216 G G(64)D(5)E(23). 0.19 gnizable in PDB entry 3na0. It is shown in Fig. 8. The right panel continued in next column shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table

6 Fig. 9. Another possible active surface on the chain 3na0A. The larger cluster Fig. 10. Another possible active surface on the chain 3na0A. The larger it belongs to is shown in blue. cluster it belongs to is shown in blue.

Table 10. continued Table 12. continued res type substitutions(%) cvg res type substitutions(%) cvg antn N(4)S 437 P P(99)R 0.02 217 E E(55)T(8)K(20) 0.19 355 E E(91)Q(7). 0.03 S(7).Q(5)VNR 382 K K(96)R(3) 0.03 222 L L(70)V(3)T(3) 0.21 439 R R(98)SM 0.03 F(16).M(3)CI(1) 118 P P(97).(1)G 0.04 215 F Y(38)F(49)L(6) 0.22 120 R R(97).(1)E 0.04 site S(3).K 271 D D(96)Q(1)GR 0.04 440 W W(97)SFE 0.04 Table 10. Residues forming surface ”patch” in 3na0A. 455 F F(94)WL.(3) 0.05 458 G G(96).(3) 0.05 330 T T(94)S(4)A 0.06 Table 11. 460 R R(94).(4)P 0.06 site res type disruptive 462 C C(94)S.(4) 0.06 site mutations 464 G G(94)R.(4) 0.06 site 218 R (T)(D)(S)(YVCAG) 337 W M(18)W(66)F(12) 0.07 220 G (R)(K)(H)(FEW) YK 177 D (R)(FWH)(Y)(CG) 376 L L(75)FY(22)M 0.07 216 G (R)(FWH)(K)(E) 456 G G(94)W.(3)S 0.07 site 217 E (FW)(H)(Y)(R) 346 L P(83)L(13)QMSR 0.08 222 L (R)(Y)(H)(K) 385 L L(85)M(8)F(2)IQ 0.08 site 215 F (KE)(T)(D)(Q) ES 114 E D(5)E(92).(1) 0.09 Table 11. Disruptive mutations for the surface patch in 3na0A. 130 H R(82).(2)H(13)L 0.09 K 429 F F(90)Y(5)TM(1)H 0.09 Another group of surface residues is shown in Fig.10. The right panel 466 R R(94).(4)F 0.09 shows (in blue) the rest of the larger cluster this surface belongs to. 468 A A(94).(4)P 0.09 The residues belonging to this surface ”patch” are listed in Table 12, 331 T T(83)V(9)A(3)I 0.10 while Table 13 suggests possible disruptive replacements for these S(2) residues (see Section 3.6). 375 P P(91)AKR(2)H(2) 0.10 Table 12. S(1) res type substitutions(%) cvg antn 379 A A(72)NC(2)G(20) 0.11 151 R R(100) 0.01 S(4) 349 Q Q(99). 0.01 413 T T(94)S(1)V(2)MI 0.11 386 R R(100) 0.01 323 M L(74)M(19)V(3) 0.12 site 389 P P(100) 0.01 I(1)G 396 R R(100) 0.01 site 334 T P(22)T(59)V 0.12 site 410 P P(100) 0.01 continued in next column 147 W W(97).(1)V 0.02 site continued in next column

7 Table 12. continued Table 12. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn S(13)GA(1)D 454 G A(22)P(42)G(27) 0.24 site 387 L L(92)V(2)M(3)IF 0.12 VS(1)T(2).(3) 465 R R(87)K(7)T.(4) 0.12 315 D T(1)D(30)E(33) 0.25 159 M L(54)M(35)F(2) 0.13 S(6)A(16)L(5) I(6)H N(2)RV 327 G S(19)G(66)A(14) 0.13 site 319 N N(55)T(2)S(27)D 0.25 378 K R(32)K(66)W 0.13 A(10)LHM(1) 115 G S(27)G(68).(1)R 0.14 341 E E(66)H(5)Q(5) 0.25 P L(1)N(12)AT(2)V 126 W W(83)M(3).(2)D 0.14 MIC(2) I(2)L(5)F(2) 305 L L(70)M(5)F(5) 0.14 Table 12. Residues forming surface ”patch” in 3na0A. I(10)V(6)Y(2) 144 S G(75).(2)D(7) 0.15 S(12)AR Table 13. 270 W W(88)QS(4)GFR 0.15 res type disruptive M(1)LIH mutations 340 Y F(29)Y(66)H(1) 0.15 151 R (TD)(SYEVCLAPIG)(FMW)(N) L(1)S 349 Q (Y)(FTWH)(SVCAG)(D) 401 D D(82)E(11)A(4) 0.15 386 R (TD)(SYEVCLAPIG)(FMW)(N) P(2) 389 P (YR)(TH)(SKECG)(FQWD) 155 N N(71)S(4)A(5) 0.16 396 R (TD)(SYEVCLAPIG)(FMW)(N) R(1)Q(10)D(5)G 410 P (YR)(TH)(SKECG)(FQWD) 471 E E(82)Q(10).(5)N 0.16 147 W (E)(K)(D)(Q) K 437 P (Y)(T)(SECHRG)(D) 318 A A(72)G(17)V(4) 0.17 355 E (FWH)(YVCAG)(TR)(S) S(1)M(1)LI 382 K (Y)(T)(FW)(SVCAG) 322 E D(13)E(83)GPK 0.17 439 R (TYD)(CG)(E)(SFVLAWPI) 326 G G(69)A(28).H 0.18 site 118 P (R)(Y)(H)(TKE) 343 A A(64)S(19)G(14) 0.18 120 R (T)(VCAG)(Y)(SLPDI) CI 271 D (FW)(HR)(Y)(VA) 432 P P(83)A(6)S(8)G 0.19 440 W (K)(E)(Q)(TR) 390 I V(65)I(22)S(8) 0.20 site 455 F (KE)(T)(QD)(SCRG) G(1)T(1)A 458 G (KER)(FQMWHD)(NLPI)(Y) 461 Q Q(54)S(10)A(9) 0.20 330 T (KR)(QH)(FMW)(E) M(19)G(1)R.(4) 460 R (T)(D)(CG)(Y) 335 L L(80)M(9)TI(5)G 0.21 462 C (KR)(E)(FMWH)(Q) DVS 464 G (E)(D)(KM)(FW) 344 R R(76)K(15)Q(2) 0.21 337 W (E)(K)(T)(D) H(3)NAW 376 L (R)(TY)(K)(EH) 411 A A(66)K(21)Q(3) 0.21 456 G (K)(ER)(Q)(MD) S(2)P(1)V(2) 346 L (Y)(R)(TH)(CG) C(1)R 385 L (Y)(R)(H)(T) 423 G G(73)T(1)S(15) 0.22 114 E (FW)(H)(VCAG)(R) C(1)YA(5).D 130 H (TE)(D)(SCG)(VMA) 469 E E(88)Q(3).(5)GV 0.23 429 F (K)(E)(Q)(D) 139 V V(67)L(20)I(1) 0.24 466 R (TD)(SECG)(VLAPI)(Y) P(6).(2)E 468 A (Y)(R)(KE)(H) 311 M M(12)V(4)L(80)F 0.24 331 T (R)(K)(H)(Q) IG 375 P (Y)(T)(R)(E) 359 A A(57)V(28)G(1) 0.24 379 A (R)(KE)(Y)(H) N(1)HI(1)T.S(5) continued in next column continued in next column

8 Table 13. continued 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 413 T (R)(K)(H)(FQW) may be interchangeable at that position in the protein, so if one wants 323 M (Y)(H)(R)(T) to affect the protein by a point mutation, they should be avoided. For 334 T (R)(K)(H)(Q) example if the substitutions are “RVK” and the original protein has 387 L (R)(Y)(T)(H) an R at that position, it is advisable to try anything, but RVK. Conver- 465 R (D)(T)(YVLAPI)(SFECWG) sely, when looking for substitutions which will not affect the protein, 159 M (Y)(T)(R)(S) one may try replacing, R with K, or (perhaps more surprisingly), with 327 G (KR)(E)(QH)(FMW) V. The percentage of times the substitution appears in the alignment 378 K (TY)(SCG)(FVAWD)(E) is given in the immediately following bracket. No percentage is given 115 G (E)(KR)(FWHD)(QM) in the cases when it is smaller than 1%. This is meant to be a rough 126 W (K)(T)(E)(R) guide - due to rounding errors these percentages often do not add up 305 L (R)(Y)(T)(KH) to 100%. 144 S (KR)(FWH)(M)(Q) 270 W (E)(K)(T)(D) 3.3 Surface 340 Y (K)(Q)(EM)(R) To detect candidates for novel functional interfaces, first we look for 401 D (R)(H)(FYW)(K) residues that are solvent accessible (according to DSSP program) by 2 155 N (Y)(FWH)(T)(R) at least 10A˚ , which is roughly the area needed for one water mole- 471 E (FW)(H)(Y)(VCAG) cule to come in the contact with the residue. Furthermore, we require 318 A (R)(Y)(K)(E) that these residues form a “cluster” of residues which have neighbor 322 E (FWH)(Y)(R)(VCAG) within 5A˚ from any of their heavy atoms. 326 G (E)(K)(R)(QD) Note, however, that, if our picture of protein evolution is correct, 343 A (R)(K)(E)(Y) the neighboring residues which are not surface accessible might be 432 P (R)(Y)(H)(K) equally important in maintaining the interaction specificity - they 390 I (R)(Y)(H)(K) should not be automatically dropped from consideration when choo- 461 Q (Y)(H)(FW)(T) sing the set for mutagenesis. (Especially if they form a cluster with 335 L (R)(Y)(H)(K) the surface residues.) 344 R (T)(D)(Y)(E) 411 A (Y)(E)(R)(KH) 3.4 Number of contacts 423 G (KR)(E)(Q)(MH) Another column worth noting is denoted “noc/bb”; it tells the num- 469 E (H)(FW)(Y)(R) ber of contacts heavy atoms of the residue in question make across 139 V (YR)(KH)(E)(Q) the interface, as well as how many of them are realized through the 311 M (Y)(HR)(T)(S) backbone atoms (if all or most contacts are through the backbone, 359 A (R)(K)(E)(Y) mutation presumably won’t have strong impact). Two heavy atoms 454 G (R)(K)(E)(H) are considered to be “in contact” if their centers are closer than 5A˚ . 315 D (R)(H)(FW)(Y) 319 N (Y)(H)(R)(FW) 3.5 Annotation 341 E (H)(Y)(FWR)(CG) If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” Table 13. Disruptive mutations for the surface patch in 3na0A. appears. Annotations carried over from PDB are the following: site (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

9 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. 11. 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. 11. the scripts and launch the viewer.)

10 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 • 3na0A.complex.pdb - coordinates of 3na0A with all of its visit: interacting partners http://mammoth.bcm.tmc.edu/traceview/ • 3na0A.etvx - ET viewer input file for 3na0A The viewer is self-unpacking and self-installing. Input files to be used • 3na0A.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 3na0A main report. • 3na0A.ranks - Ranks file in sequence order for 3na0A 4.5 Citing this work • 3na0A.clusters - Cluster descriptions for 3na0A The method used to rank residues and make predictions in this report • 3na0A.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 3na0A Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 3na0A.descr - description of sequences used in 3na0A msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 3na0A.ranks sorted - full listing of residues and their ranking of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- for 3na0A tionary Trace Method Defines Binding Surfaces Common to Protein • Families” J. Mol. Bio. 257: 342-358. 3na0A.3na0C.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 3na0A.cbcvg - used by other 3na0A – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 3na0A.3na0AHEM601.if.pml - Pymol script for Figure 6 of service for comparative analysis of .” Bioinformatics • 3na0A.3na0A2DC602.if.pml - Pymol script for Figure 7 22:1656-7. • 3na0C.complex.pdb - coordinates of 3na0C with all of its 4.6 About report maker interacting partners 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.

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