Pages 1–12 2jgu Evolutionary trace report by report maker July 23, 2010

4.3.1 Alistat 11 4.3.2 CE 11 4.3.3 DSSP 11 4.3.4 HSSP 11 4.3.5 LaTex 11 4.3.6 Muscle 11 4.3.7 Pymol 12 4.4 Note about ET Viewer 12 4.5 Citing this work 12 4.6 About report maker 12 4.7 Attachments 12

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2jgu): Title: Crystal structure of dna-directed dna polymerase Compound: Mol id: 1; molecule: dna polymerase; synonym: pfu polymerase, dna polymerase pfu; chain: a; ec: 2.7.7.7; engineered: yes Organism, scientific name: Pyrococcus Furiosus 2jgu contains a single unique chain 2jguA (712 residues long).

CONTENTS 2 CHAIN 2JGUA 1 Introduction 1 2.1 P61876 overview 2 Chain 2jguA 1 From SwissProt, id P61876, 90% identical to 2jguA: 2.1 P61876 overview 1 Description: DNA polymerase (EC 2.7.7.7) (Pwo polymerase). 2.2 Multiple sequence alignment for 2jguA 1 Organism, scientific name: Pyrococcus woesei. 2.3 Residue ranking in 2jguA 1 Taxonomy: Archaea; Euryarchaeota; Thermococci; Thermococca- 2.4 Top ranking residues in 2jguA and their position on les; Thermococcaceae; Pyrococcus. the structure 2 Function: In addition to polymerase activity, this DNA polymerase 2.4.1 Clustering of residues at 25% coverage. 2 exhibits 3’ to 5’ exonuclease activity. 2.4.2 Overlap with known functional surfaces at Catalytic activity: Deoxynucleoside triphosphate + DNA(n) = 25% coverage. 3 diphosphate + DNA(n+1). 2.4.3 Possible novel functional surfaces at 25% Subunit: Monomer. coverage. 4 Similarity: Belongs to the DNA polymerase type-B family. About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 10 collaboration between the Swiss Institute of Bioinformatics and the 3.1 Coverage 10 EMBL outstation - the European Bioinformatics Institute. There are 3.2 Known substitutions 10 no restrictions on its use as long as its content is in no way modified 3.3 Surface 10 and this statement is not removed. 3.4 Number of contacts 10 3.5 Annotation 10 2.2 Multiple sequence alignment for 2jguA 3.6 Mutation suggestions 11 For the chain 2jguA, the alignment 2jguA.msf (attached) with 79 sequences was used. The alignment was assembled through combi- 4 Appendix 11 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 11 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 11 report, under the name of 2jguA.msf. Its statistics, from the alistat 4.3 Credits 11 program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-237 in 2jguA colored by their relative importance. (See Fig. 3. Residues 475-758 in 2jguA colored by their relative importance. (See Appendix, Fig.13, for the coloring scheme.) Appendix, Fig.13, for the coloring scheme.)

2.4 Top ranking residues in 2jguA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 4 shows residues in 2jguA 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. 2. Residues 238-474 in 2jguA colored by their relative importance. (See Appendix, Fig.13, for the coloring scheme.)

Format: MSF Number of sequences: 79 Total number of residues: 53686 Smallest: 566 Largest: 712 Average length: 679.6 Alignment length: 712 Average identity: 29% Most related pair: 99% Most unrelated pair: 17% Most distant seq: 32%

Furthermore, 1% of residues show as conserved in this alignment. The alignment consists of 31% eukaryotic ( 11% vertebrata, 11% Fig. 4. Residues in 2jguA, colored by their relative importance. Clockwise: fungi, 2% plantae), 12% prokaryotic, 39% archaean, and 17% viral front, back, top and bottom views. sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 2jguA.descr. 2.4.1 Clustering of residues at 25% coverage. Fig. 5 shows the top 25% of all residues, this time colored according to clusters they 2.3 Residue ranking in 2jguA belong to. The clusters in Fig.5 are composed of the residues listed The 2jguA sequence is shown in Figs. 1–3, with each residue colored in Table 1. according to its estimated importance. The full listing of residues in 2jguA can be found in the file called 2jguA.ranks sorted in the attachment.

2 Table 1. continued cluster size member color residues yellow 8 679,680,681,683,684,700,702 703 green 7 617,619,621,661,729,732,733 purple 5 19,203,204,254,255 azure 2 343,345

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

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. Manganese (ii) ion binding site. Table 2 lists the top 25% of resi- dues at the interface with 2jguAMN1758 (manganese (ii) ion). 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 antn (%) bb (A˚ ) Fig. 5. Residues in 2jguA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for 343 D D(26) 0.20 3/0 3.39 site the coloring scheme). Clockwise: front, back, top and bottom views. The Q(6) corresponding Pymol script is attached. C(5) E(7) Y(13) Table 1. R(34) cluster size member F(6) color residues red 120 37,39,111,113,114,119,123 Table 2. The top 25% of residues in 2jguA at the interface with manga- 329,332,348,349,350,352,353 nese (ii) ion.(Field names: res: residue number in the PDB entry; type: amino 356,357,361,368,369,385,387 acid type; substs: substitutions seen in the alignment; with the percentage of 388,390,393,396,397,398,401 each type in the bracket; noc/bb: number of contacts with the ligand, with 403,404,405,406,408,409,410 the number of contacts realized through backbone atoms given in the bracket; 411,412,413,414,418,419,420 dist: distance of closest apporach to the ligand. ) 421,422,423,448,450,454,457 458,461,465,480,481,482,483 484,485,486,487,488,490,491 Table 3. 492,493,494,495,496,497,498 res type disruptive 499,503,504,505,506,507,510 mutations 511,512,513,514,515,518,519 343 D (R)(FW)(H)(VA) 522,537,539,540,541,542,543 544,546,569,572,575,576,578 Table 3. List of disruptive mutations for the top 25% of residues in 2jguA, 579,581,582,584,587,588,589 that are at the interface with manganese (ii) ion. 590,591,592,593,594,595,596 597,607,608,609,610,632,744 Figure 6 shows residues in 2jguA colored by their importance, at the 745 interface with 2jguAMN1758. blue 33 140,141,142,143,144,187,191 Manganese (ii) ion binding site. Table 4 lists the top 25% of resi- 194,207,208,209,210,214,215 dues at the interface with 2jguAMN1759 (manganese (ii) ion). The 217,218,222,223,259,260,261 following table (Table 5) suggests possible disruptive replacements 270,273,275,278,283,287,308 for these residues (see Section 3.6). 311,315,316,319,322 Table 4. continued in next column res type subst’s cvg noc/ dist antn (%) bb (A˚ ) continued in next column

3 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 5. res type disruptive mutations 315 D (R)(FWH)(Y)(VCAG) 143 E (FWH)(Y)(R)(CG) 141 D (R)(H)(FW)(K) 311 Y (K)(EQ)(M)(R) 142 I (R)(Y)(H)(KE)

Table 5. List of disruptive mutations for the top 25% of residues in 2jguA, that are at the interface with manganese (ii) ion.

Fig. 6. Residues in 2jguA, at the interface with manganese (ii) ion, colored by their relative importance. The ligand (manganese (ii) ion) 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 2jguA.)

Table 4. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 315 D D(98) 0.03 4/0 2.03 N(1) 143 E E(88) 0.05 4/0 2.14 site K(8) S(1) V(1) 141 D D(87) 0.10 4/0 2.23 site A(2) S(7) G(1) Fig. 7. Residues in 2jguA, at the interface with manganese (ii) ion, colored N(1) by their relative importance. The ligand (manganese (ii) ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well 311 Y Y(87) 0.10 3/0 4.22 as on the line of sight to the ligand were removed. (See Appendix for the H(8) coloring scheme for the protein chain 2jguA.) L(1) N(1) W(1) Figure 7 shows residues in 2jguA colored by their importance, at the 142 I I(84) 0.17 3/3 3.31 site interface with 2jguAMN1759. F(2) 2.4.3 Possible novel functional surfaces at 25% coverage. One M(6) group of residues is conserved on the 2jguA surface, away from (or L(2) susbtantially larger than) other functional sites and interfaces reco- C(1) gnizable in PDB entry 2jgu. It is shown in Fig. 8. The residues V(1) belonging to this surface ”patch” are listed in Table 6, while Table G(1) 7 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. The top 25% of residues in 2jguA at the interface with manga- nese (ii) ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of

4 Fig. 9. Another possible active surface on the chain 2jguA. The larger cluster it belongs to is shown in blue.

Fig. 8. A possible active surface on the chain 2jguA.

Table 6. res type substitutions(%) cvg 204 D D(70)E(15)P(13) 0.08 254 G G(84)A(7)H(2) 0.08 R(1)P(1)E(1) Y(1) 203 P P(68).(16)V(12) 0.11 A(1)I(1) 19 F F(48)W(17)Y(17) 0.19 L(13)I(1)V(1) 255 R R(65)V(6)G(12) 0.23 I(8)S(2)L(2) F(1)

Table 6. Residues forming surface ”patch” in 2jguA.

Table 7. res type disruptive mutations 204 D (R)(H)(FW)(Y) 254 G (K)(E)(R)(Q) 203 P (R)(Y)(H)(T) 19 F (K)(E)(Q)(D) 255 R (D)(E)(TY)(S)

Table 7. Disruptive mutations for the surface patch in 2jguA.

Another group of surface residues is shown in Fig.9. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 8, while Table 9 suggests possible disruptive replacements for these residues (see Section 3.6).

5 Table 8. Table 9. res type substitutions(%) cvg antn res type disruptive 259 D D(100) 0.01 mutations 275 L L(100) 0.01 259 D (R)(FWH)(KYVCAG)(TQM) 315 D D(98)N(1) 0.03 275 L (YR)(TH)(SKECG)(FQWD) 210 N N(96)D(3) 0.04 315 D (R)(FWH)(Y)(VCAG) 222 R R(92)H(7) 0.04 210 N (Y)(FWH)(TR)(VCAG) 143 E E(88)K(8)S(1) 0.05 site 222 R (TD)(E)(SVCLAPIG)(YM) V(1) 143 E (FWH)(Y)(R)(CG) 209 Y W(12)Y(64)F(11) 0.05 209 Y (K)(Q)(E)(M) H(11) 214 F (K)(E)(QD)(R) 214 F F(93)G(3)Y(1) 0.05 187 E (FW)(H)(YVCAG)(T) M(1) 287 K (Y)(FTW)(CG)(SVA) 187 E E(86)D(6)R(7) 0.06 141 D (R)(H)(FW)(K) 287 K K(79)R(17)E(1) 0.06 311 Y (K)(EQ)(M)(R) A(1) 215 D (R)(H)(FW)(Y) 141 D D(87)A(2)S(7) 0.10 site 273 Y (K)(Q)(R)(EM) G(1)N(1) 218 Y (K)(QR)(E)(NM) 311 Y Y(87)H(8)L(1) 0.10 223 A (R)(Y)(K)(E) N(1)W(1) 194 F (K)(E)(QR)(D) 215 D D(91)I(1)E(3) 0.11 319 T (R)(K)(H)(Q) M(2).(1) 144 T (R)(K)(H)(FW) 273 Y Y(75)K(7)F(11) 0.13 308 V (R)(Y)(K)(E) L(1)E(1)W(2) 217 P (Y)(R)(H)(T) 218 Y Y(75)M(5)L(8) 0.14 270 L (R)(Y)(H)(K) V(8)F(1) 261 Y (K)(Q)(ER)(M) 223 A L(5)A(73)S(3) 0.14 140 F (KE)(R)(Q)(D) M(6)I(10)C(1) 194 F F(58)W(20)A(3) 0.16 Table 9. Disruptive mutations for the surface patch in 2jguA. L(13)V(1)I(1) T(1) Another group of surface residues is shown in Fig.10. The right panel 319 T C(2)A(3)T(36) 0.17 shows (in blue) the rest of the larger cluster this surface belongs to. V(27)P(20)I(7) S(1) 144 T V(26)T(27)C(40) 0.18 M(1)N(1)G(1) L(1) 308 V L(69)V(18)T(2) 0.18 I(7)M(1) 217 P P(64)A(6)K(6) 0.20 R(11)D(3)E(7) 270 L L(49)V(3)A(5) 0.20 F(24)E(3)C(6) S(2)I(2)P(1) T(1) 261 Y L(21)Y(49)I(10) 0.23 F(7)E(6)H(1) Fig. 10. Another possible active surface on the chain 2jguA. The larger A(1)W(1)K(1) cluster it belongs to is shown in blue. 140 F F(75)I(11)L(10) 0.25 G(1)V(1) The residues belonging to this surface ”patch” are listed in Table 10, while Table 11 suggests possible disruptive replacements for these Table 8. Residues forming surface ”patch” in 2jguA. residues (see Section 3.6). Table 10. res type substitutions(%) cvg antn 369 P P(100) 0.01 405 D D(100) 0.01 continued in next column

6 Table 10. continued Table 10. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn 410 Y Y(100) 0.01 497 Y V(32)Y(22)F(30) 0.09 411 P P(100) 0.01 C(11)L(2) 492 N N(100) 0.01 522 I I(58)L(31)M(10) 0.09 495 Y Y(100) 0.01 512 E A(20)E(48)S(25) 0.10 539 Y Y(100) 0.01 G(1)Q(5) 593 K K(100) 0.01 594 R R(60)K(22)N(13) 0.10 595 Y Y(100) 0.01 Q(1)H(1) 519 R R(89)H(10) 0.02 491 A M(13)A(50)C(17) 0.11 542 T T(98)S(1) 0.02 I(12)L(3)T(1) 543 D D(98).(1) 0.02 119 R R(74)N(12)L(7) 0.12 592 K K(98)E(1) 0.02 S(2)K(1)Q(1) 608 G G(98).(1) 0.02 350 G A(7)G(18)Q(35) 0.12 387 G G(98)E(1) 0.03 S(27)V(10) 390 V V(98)I(1) 0.03 465 K K(74)Y(13)R(10) 0.12 396 G G(98)D(1) 0.03 E(1) 461 R R(98)I(1) 0.03 493 S S(59)A(39)T(1) 0.12 488 K K(98)R(1) 0.03 546 Y M(34)F(55)H(1) 0.12 496 G G(98)D(1) 0.03 Y(7).(1) 393 P P(86)S(10)I(1) 0.04 576 L F(5)L(46)I(40) 0.12 A(2) V(3)M(2).(1) 408 S S(97)T(1)A(1) 0.04 332 Q E(58)Q(24)L(10) 0.13 484 Q Q(86)S(2).(10) 0.04 V(2)M(3)Y(1) L(1) 457 L L(70)W(17)I(10) 0.13 515 T T(97)F(1)A(1) 0.04 F(1) 541 D D(98)X(1) 0.04 352 L D(2)R(30)L(11) 0.14 518 G G(97)A(1)N(1) 0.05 K(25)W(13)A(10) 418 N N(91)L(7)R(1) 0.06 Q(1)Y(1)M(2) 421 P P(35)Y(53)F(11) 0.06 I(1) 579 E E(84)K(6)D(7) 0.06 385 Y Y(78)F(10)S(11) 0.14 .(1) 507 C D(12)S(6)C(55) 0.14 S-S 388 G G(44)A(55) 0.07 A(20)F(2)V(1) 409 L L(89)M(7)I(1) 0.07 N(1) S(1) 511 A G(2)A(78)S(17) 0.14 448 G G(68)S(17)H(10) 0.07 V(1) A(3) 37 Y Y(78)V(8)E(3) 0.15 499 G G(93)A(5)R(1) 0.07 T(5)I(1).(1) 537 V V(96)I(2)P(1) 0.07 R(1) 581 E E(65)D(31)N(1) 0.07 329 M F(17)M(12)E(17) 0.15 .(1) L(27)N(20)Y(1) 607 R T(2)V(6)R(11) 0.07 D(1)V(1) K(78).(1) 398 W R(2)H(12)W(10) 0.15 505 W L(49)W(22)F(27) 0.08 Y(65)F(7)Q(1) 506 Y Y(45)P(37)F(11) 0.08 572 L Y(13)F(50)L(17) 0.15 H(3)T(1) H(3)W(10).(3) 540 I G(86)A(8)I(5) 0.08 403 Y V(51)Y(7)T(20) 0.16 111 E E(75)D(10)G(10) 0.09 L(11)M(1)C(2) A(1)N(1)Y(1) S(5) 123 D D(78)E(11)K(3) 0.09 480 L F(1)L(63)Y(22) 0.16 N(3)S(2) .(10)I(1)M(1) 404 L L(77)F(5)M(3) 0.09 483 R Q(15)R(48)K(7) 0.16 V(13) V(11)I(2).(10) 406 Y L(2)F(86)Y(11) 0.09 E(3)L(1) continued in next column continued in next column

7 Table 10. continued Table 10. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn 584 Y Y(58)L(5)F(35) 0.16 M(1)G(1)L(1) M(1) 610 E E(55)D(24)L(7) 0.22 589 F Q(3)F(30)L(50) 0.16 A(5)T(2)V(3) M(11)S(1)Y(1) .(1) I(1) 632 L V(2)L(65)F(15) 0.22 597 V G(73)V(7)X(1) 0.16 T(2)S(3)R(1) A(11)L(5).(1) I(1).(1)M(2) 39 Y Y(73)L(10)F(11) 0.17 A(2)G(1) I(1).(1)G(1) 114 I I(60)V(27)Q(3) 0.23 M(1) T(6)L(1) 113 D D(44)H(7)N(43) 0.17 590 V A(2)T(12)V(15) 0.23 R(1)E(2)S(1) I(31)S(13)L(20) 348 S G(64)S(18)E(7) 0.17 Q(2)H(1) A(3)Q(3)N(1) 356 F .(2)L(41)F(7) 0.24 458 L L(49)I(11)R(13) 0.17 C(16)Q(20)R(2) W(10)V(11)S(1) M(1)I(1)Y(5) M(1)Y(1) S(1) 490 L I(15)M(2)L(18) 0.18 397 L V(11)I(12)L(27) 0.24 T(29)F(8)S(20) Y(21)F(26) V(2)Y(2) 485 K A(5)R(20)L(37) 0.24 504 R R(48)L(11)P(12) 0.18 Q(5).(10)K(16) K(17)I(5)S(1) S(2)N(1)M(1) Q(2)M(1) 588 F F(21)I(10)A(7) 0.24 349 T D(2)V(15)T(11) 0.19 L(51)V(2)M(5) Q(37)S(13)R(12) T(1) I(3)Y(1)F(1) 745 I I(75)A(5)L(12) 0.24 481 D D(65)N(15)T(2) 0.19 M(1)V(2)C(1) .(10)E(6) .(1) 582 G K(60)G(22)T(8) 0.20 525 V T(67)S(13)A(7) 0.25 A(1)E(1).(1) I(3)V(7) D(1)N(1)I(1) 591 T G(26)E(7)T(22) 0.25 744 R R(50)N(17)Q(13) 0.20 C(5)N(16)A(2) S(5)G(7)A(2) K(11)S(7) K(1)H(1) 482 Y R(2)A(3)Y(11) 0.21 Table 10. Residues forming surface ”patch” in 2jguA. K(17)V(13)G(21) .(10)I(12)E(3) H(1)T(1) Table 11. 487 I V(2)L(50)I(34) 0.21 res type disruptive M(11)Y(1) mutations 569 N N(37)S(26)T(16) 0.21 369 P (YR)(TH)(SKECG)(FQWD) K(10)E(6)A(1) 405 D (R)(FWH)(KYVCAG)(TQM) H(1) 410 Y (K)(QM)(NEVLAPIR)(D) 575 L R(2).(21)L(17) 0.21 411 P (YR)(TH)(SKECG)(FQWD) P(39)A(8)C(3) 492 N (Y)(FTWH)(SEVCARG)(MD) G(2)N(1)Y(1) 495 Y (K)(QM)(NEVLAPIR)(D) E(1) 539 Y (K)(QM)(NEVLAPIR)(D) 609 L F(7)L(46)V(17) 0.21 593 K (Y)(FTW)(SVCAG)(HD) M(10)I(15)Y(1) 595 Y (K)(QM)(NEVLAPIR)(D) .(1) 519 R (TD)(E)(SVCLAPIG)(YM) 422 D E(18)D(18)S(18) 0.22 542 T (KR)(FQMWH)(NELPI)(D) T(29)V(7)C(2) 543 D (R)(FWH)(VCAG)(KY) continued in next column 592 K (Y)(FW)(T)(VCAG) continued in next column

8 Table 11. continued Table 11. continued res type disruptive res type disruptive mutations mutations 608 G (KER)(FQMWHD)(NLPI)(Y) 480 L (R)(Y)(T)(H) 387 G (R)(FKWH)(YEQM)(NLPDI) 483 R (T)(Y)(D)(CG) 390 V (YR)(KE)(H)(QD) 584 Y (K)(QR)(E)(NM) 396 G (R)(K)(FWH)(EQM) 589 F (K)(E)(T)(R) 461 R (T)(YD)(SECG)(VA) 597 V (R)(KYE)(H)(QD) 488 K (Y)(T)(FW)(SVCAG) 39 Y (K)(Q)(R)(E) 496 G (R)(K)(FWH)(EQM) 113 D (R)(FW)(H)(Y) 393 P (R)(Y)(H)(K) 348 S (R)(H)(FW)(K) 408 S (KR)(QH)(FMW)(E) 458 L (R)(Y)(T)(H) 484 Q (Y)(H)(FW)(T) 490 L (R)(Y)(KH)(E) 515 T (K)(R)(Q)(E) 504 R (TY)(D)(CG)(S) 541 D (R)(FWH)(KY)(VCAG) 349 T (KR)(H)(Q)(M) 518 G (E)(R)(K)(H) 481 D (R)(FWH)(VA)(Y) 418 N (Y)(T)(FWH)(SECG) 582 G (R)(H)(FW)(K) 421 P (R)(K)(TE)(Y) 744 R (D)(TY)(E)(SFVCLAWPIG) 579 E (FW)(H)(YVCAG)(R) 482 Y (K)(Q)(M)(E) 388 G (KER)(QHD)(FYMW)(N) 487 I (R)(Y)(H)(T) 409 L (YR)(H)(T)(K) 569 N (Y)(FW)(H)(TR) 448 G (KE)(R)(Q)(MD) 575 L (R)(Y)(H)(T) 499 G (E)(D)(K)(R) 609 L (R)(Y)(T)(H) 537 V (YR)(KE)(H)(QD) 422 D (R)(H)(FW)(K) 581 E (FWH)(YVCARG)(T)(S) 610 E (H)(R)(FW)(Y) 607 R (D)(TY)(E)(LPI) 632 L (R)(Y)(H)(KE) 505 W (KE)(T)(QD)(R) 114 I (R)(Y)(H)(T) 506 Y (K)(Q)(EM)(R) 590 V (R)(Y)(KE)(H) 540 I (R)(Y)(H)(KE) 356 F (E)(K)(TD)(Q) 111 E (H)(R)(FW)(Y) 397 L (R)(Y)(K)(E) 123 D (FW)(R)(H)(Y) 485 K (Y)(T)(FW)(CG) 404 L (YR)(T)(H)(KE) 588 F (K)(E)(R)(QD) 406 Y (K)(Q)(R)(E) 745 I (Y)(R)(H)(T) 497 Y (K)(Q)(E)(R) 525 V (R)(K)(YE)(H) 522 I (Y)(R)(TH)(CG) 591 T (R)(K)(FWH)(M) 512 E (H)(FW)(R)(Y) 594 R (T)(YD)(VCAG)(S) Table 11. Disruptive mutations for the surface patch in 2jguA. 491 A (R)(Y)(K)(E) 119 R (TY)(D)(CG)(SE) Another group of surface residues is shown in Fig.11. The residues 350 G (R)(KE)(H)(YD) belonging to this surface ”patch” are listed in Table 12, while Table 465 K (Y)(FTW)(VA)(CG) 13 suggests possible disruptive replacements for these residues (see 493 S (KR)(QH)(FMW)(E) Section 3.6). 546 Y (K)(Q)(EM)(N) 576 L (YR)(T)(H)(KE) Table 12. 332 Q (Y)(H)(T)(FW) res type substitutions(%) cvg 457 L (R)(TY)(KE)(SCHG) 680 H Q(3)H(93)N(1) 0.04 352 L (Y)(R)(T)(H) .(1) 385 Y (K)(Q)(M)(ER) 702 Y R(2)Y(84)F(7) 0.09 507 C (R)(K)(E)(H) I(1)W(2)K(1) 511 A (KR)(E)(Y)(QH) 703 I L(2)V(77)I(18) 0.14 37 Y (K)(Q)(M)(R) S(1) 329 M (Y)(H)(TR)(CG) 679 P A(22)P(60)Q(13) 0.15 398 W (E)(K)(TD)(Q) .(1)S(1) 572 L (R)(T)(K)(E) 683 V G(2)A(35)V(39) 0.15 403 Y (K)(R)(Q)(M) L(20)I(1).(1) continued in next column 684 A A(77)I(2)Y(11) 0.15 continued in next column

9 Fig. 11. Another possible active surface on the chain 2jguA. Fig. 12. Another possible active surface on the chain 2jguA.

Table 12. continued 15 suggests possible disruptive replacements for these residues (see res type substitutions(%) cvg Section 3.6). V(3).(1)F(1) Table 14. G(2) res type substitutions(%) cvg 700 I P(2)I(63)V(31) 0.21 733 Y M(2)Y(94)C(1) 0.05 W(1)T(1) .(1) 681 V V(67)L(16)A(8) 0.25 617 S A(3)C(54)S(15) 0.11 I(6).(1) P(13)T(10)F(1) 698 M S(5)V(2)T(6) 0.25 .(1) D(62)G(10)N(3) 619 I I(16)L(50)F(30) 0.11 A(1)M(6)E(1) V(1).(1) R(1) 729 D D(78)H(2)A(12) 0.13 E(1)N(2)V(1) Table 12. Residues forming surface ”patch” in 2jguA. .(1) 621 K K(56)S(6)Q(29) 0.14 A(5).(1)R(1) Table 13. 661 L V(2)L(82)F(8) 0.17 res type disruptive Y(3).(1)I(1) mutations 732 Y K(21)Y(48)H(13) 0.22 680 H (TE)(D)(SVMCAG)(QLPI) L(10)W(3)V(1) 702 Y (K)(E)(Q)(M) .(1) 703 I (R)(Y)(H)(K) 679 P (Y)(R)(H)(T) Table 14. Residues forming surface ”patch” in 2jguA. 683 V (R)(Y)(KE)(H) 684 A (K)(R)(E)(Q) 700 I (R)(Y)(K)(H) Table 15. 681 V (R)(Y)(KE)(H) res type disruptive 698 M (Y)(H)(R)(T) mutations 733 Y (K)(Q)(MR)(NE) Table 13. Disruptive mutations for the surface patch in 2jguA. 617 S (KR)(Q)(H)(M) 619 I (R)(Y)(T)(H) continued in next column Another group of surface residues is shown in Fig.12. The residues belonging to this surface ”patch” are listed in Table 14, while Table

10 Table 15. continued mutation presumably won’t have strong impact). Two heavy atoms res type disruptive are considered to be “in contact” if their centers are closer than 5A˚ . mutations 729 D (R)(H)(FW)(Y) 3.5 Annotation 621 K (Y)(FW)(T)(CG) If the residue annotation is available (either from the pdb file or 661 L (R)(Y)(K)(T) from other sources), another column, with the header “annotation” 732 Y (K)(Q)(E)(M) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 15. Disruptive mutations for the surface patch in 2jguA. 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- 3.1 Coverage mentarity with the substitutions found in the alignment. Note that 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 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For 4 APPENDIX example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- 4.1 File formats sely, when looking for substitutions which will not affect the protein, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment • is given in the immediately following bracket. No percentage is given alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • residue# residue number in the PDB file guide - due to rounding errors these percentages often do not add up • type amino acid type to 100%. • rank rank of the position according to older version of ET 3.3 Surface • variability has two subfields: To detect candidates for novel functional interfaces, first we look for 1. number of different amino acids appearing in in this column residues that are solvent accessible (according to DSSP program) by of the alignment 2 at least 10A˚ , which is roughly the area needed for one water mole- 2. their type cule to come in the contact with the residue. Furthermore, we require • rho ET score - the smaller this value, the lesser variability of that these residues form a “cluster” of residues which have neighbor this position across the branches of the tree (and, presumably, within 5A˚ from any of their heavy atoms. the greater the importance for the protein) Note, however, that, if our picture of protein evolution is correct, • the neighboring residues which are not surface accessible might be cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller should not be automatically dropped from consideration when choo- • gaps percentage of gaps in this column sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4.2 Color schemes used The following color scheme is used in figures with residues colored 3.4 Number of contacts by cluster size: black is a single-residue cluster; clusters composed of Another column worth noting is denoted “noc/bb”; it tells the num- more than one residue colored according to this hierarchy (ordered ber of contacts heavy atoms of the residue in question make across by descending size): red, blue, yellow, green, purple, azure, tur- the interface, as well as how many of them are realized through the quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, backbone atoms (if all or most contacts are through the backbone, bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,

11 Daruvar, and C. Sander. ”The HSSP database of protein structure- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/

COVERAGE 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison-

V Wesley,” Reading, Mass. (1986). 100% 50% 30% 5% 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. http://www.drive5.com/muscle/ V

RELATIVE IMPORTANCE 4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- Fig. 13. Coloring scheme used to color residues by their relative importance. tific LLC (2005). For more information about Pymol see http://pymol.sourceforge.net/. (Note for Windows users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 13. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.2 CE To map ligand binding sites from different of service for comparative analysis of proteins.” Bioinformatics source structures, report maker uses the CE program: 22:1656-7. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , Houston. which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 4.7 Attachments Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following files should accompany this report: by [email protected] November 18,2002, • 2jguA.complex.pdb - coordinates of 2jguA with all of its inter- http://www.cmbi.kun.nl/gv/dssp/descrip.html. acting partners • 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 2jguA.etvx - ET viewer input file for 2jguA gnment as a starting point for the analysis (sequences shorter than • 2jguA.cluster report.summary - Cluster report summary for 75% of the query are taken out, however); R. Schneider, A. de 2jguA

12 • 2jguA.ranks - Ranks file in sequence order for 2jguA • 2jguA.ranks sorted - full listing of residues and their ranking for • 2jguA.clusters - Cluster descriptions for 2jguA 2jguA • • 2jguA.msf - the multiple sequence alignment used for the chain 2jguA.2jguAMN1758.if.pml - Pymol script for Figure 6 2jguA • 2jguA.cbcvg - used by other 2jguA – related pymol scripts • 2jguA.descr - description of sequences used in 2jguA msf • 2jguA.2jguAMN1759.if.pml - Pymol script for Figure 7

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