Pages 1–15 3ksc Evolutionary trace report by report maker May 12, 2010

4.3.1 Alistat 14 4.3.2 CE 14 4.3.3 DSSP 14 4.3.4 HSSP 15 4.3.5 LaTex 15 4.3.6 Muscle 15 4.3.7 Pymol 15 4.4 Note about ET Viewer 15 4.5 Citing this work 15 4.6 About report maker 15 4.7 Attachments 15

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3ksc): Title: Crystal structure of pea prolegumin, an 11s seed globulin from pisum sativum l. Compound: Mol id: 1; molecule: lega class; chain: a, b, c, d, e, f; CONTENTS synonym: prolegumin; engineered: yes Organism, scientific name: Pisum Sativum; 1 Introduction 1 3ksc contains a single unique chain 3kscE (383 residues long) and its homologues 3kscA, 3kscF, 3kscD, 3kscC, and 3kscB. 2 Chain 3kscE 1 2.1 Q41676 overview 1 2.2 Multiple sequence alignment for 3kscE 1 2.3 Residue ranking in 3kscE 1 2.4 Top ranking residues in 3kscE and their position on 2 CHAIN 3KSCE the structure 1 2.4.1 Clustering of residues at 25% coverage. 2 2.1 Q41676 overview 2.4.2 Overlap with known functional surfaces at From SwissProt, id Q41676, 78% identical to 3kscE: 25% coverage. 2 Description: Legumin A precursor. 2.4.3 Possible novel functional surfaces at 25% Organism, scientific name: narbonensis (Narbonne vetch). coverage. 10 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3 Notes on using trace results 13 eudicotyledons; ; eurosids I; ; ; Papilionoi- 3.1 Coverage 13 deae; Vicieae; Vicia. 3.2 Known substitutions 13 3.3 Surface 13 3.4 Number of contacts 14 3.5 Annotation 14 2.2 Multiple sequence alignment for 3kscE 3.6 Mutation suggestions 14 For the chain 3kscE, the alignment 3kscE.msf (attached) with 218 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 14 database, and fragments shorter than 75% of the query as well as 4.1 File formats 14 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 14 to this report, under the name of 3kscE.msf. Its statistics, from the 4.3 Credits 14 alistat program are the following:

1 Lichtarge lab 2006 residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 7-219 in 3kscE colored by their relative importance. (See Appendix, Fig.18, for the coloring scheme.)

Fig. 2. Residues 220-488 in 3kscE colored by their relative importance. (See Appendix, Fig.18, for the coloring scheme.) Fig. 3. Residues in 3kscE, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 218 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 64427 top 25% of all residues, this time colored according to clusters they Smallest: 49 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 383 in Table 1. Average length: 295.5 Alignment length: 383 Average identity: 43% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 0% color residues Most distant seq: 48% red 32 10,43,45,46,67,71,117,118 145,325,326,346,350,351,353 358,359,360,373,374,375,376 Furthermore, <1% of residues show as conserved in this ali- 378,379,380,381,416,417,435 gnment. 436,437,441 The alignment consists of 60% eukaryotic ( 60% plantae) blue 19 20,32,34,50,332,333,337,339 sequences. (Descriptions of some sequences were not readily availa- 340,341,363,365,368,369,384 ble.) The file containing the sequence descriptions can be found in 410,411,425,432 the attachment, under the name 3kscE.descr. yellow 7 84,85,86,108,109,110,318 2.3 Residue ranking in 3kscE green 7 15,393,394,395,402,403,404 purple 6 53,57,58,77,131,132 The 3kscE sequence is shown in Figs. 1–2, with each residue colored azure 5 65,151,152,153,154 according to its estimated importance. The full listing of residues turquoise 3 26,27,29 in 3kscE can be found in the file called 3kscE.ranks sorted in the brown 2 448,451 attachment. coral 2 457,460 2.4 Top ranking residues in 3kscE and their position on magenta 2 61,162 LightSalmon 2 80,129 the structure SkyBlue 2 472,476 In the following we consider residues ranking among top 25% of resi- continued in next column dues in the protein . Figure 3 shows residues in 3kscE colored by their importance: bright red and yellow indicate more conserved/important

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) V(1)LIF KHTC 332 P A(66)G 0.18 2/0 3.96 P(13) .(13) S(3) T(1)FR 131 Y Y(77)D 0.20 1/0 4.63 .(15) F(2) H(3)T

Table 2. The top 25% of residues in 3kscE at the interface with 3kscB. (Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Fig. 4. Residues in 3kscE, colored according to the cluster they belong to: Table 3. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The res type disruptive corresponding Pymol script is attached. mutations 75 G (R)(KE)(H)(FWD) 337 P (Y)(R)(H)(T) Table 1. continued 341 R (D)(E)(T)(Y) cluster size member 332 P (R)(Y)(H)(KE) color residues 131 Y (K)(Q)(M)(R)

Table 1. Clusters of top ranking residues in 3kscE. Table 3. List of disruptive mutations for the top 25% of residues in 3kscE, that are at the interface with 3kscB.

2.4.2 Overlap with known functional surfaces at 25% coverage. Figure 5 shows residues in 3kscE colored by their importance, at the The name of the ligand is composed of the source PDB identifier interface with 3kscB. and the heteroatom name used in that file. Interface with 3kscD.Table 4 lists the top 25% of residues at the Interface with 3kscB.Table 2 lists the top 25% of residues at the interface with 3kscD. The following table (Table 5) suggests possible interface with 3kscB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). disruptive replacements for these residues (see Section 3.6). Table 4. Table 2. res type subst’s cvg noc/ dist antn res type subst’s cvg noc/ dist antn (%) bb (A˚ ) (%) bb (A˚ ) 373 P P(87) 0.00 16/2 3.66 75 G G(78) 0.07 1/1 4.69 .(12)A .(21)P 416 P P(86) 0.01 3/2 3.88 337 P P(68) 0.08 9/6 4.01 .(13) R(11)S 10 C C(73) 0.02 48/20 2.92 S-S .(13) .(25)WY E(2)TAQ 399 G G(85) 0.02 37/37 3.32 LK .(12)ER 341 R S(7) 0.14 40/0 2.98 site C R(68) 375 Y W(67) 0.03 66/0 2.64 .(13) F(1) Y(4) Y(17) continued in next column continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Q(75)Y .(12) E(3)P 43 C C(78) 0.07 4/3 3.74 S-S .(19)LH 378 N N(79) 0.07 12/10 3.31 A(2) .(12) S(1) D(1)KTQ 379 A A(77) 0.09 11/10 3.81 C(2) S(5) .(12) G(1)D 476 K K(75) 0.13 26/0 2.78 R(5) .(16)TM D Fig. 5. Residues in 3kscE, at the interface with 3kscB, colored by their rela- 394 V I(19) 0.17 2/2 4.78 tive importance. 3kscB is shown in backbone representation (See Appendix V(64)A for the coloring scheme for the protein chain 3kscE.) .(12)NM S 404 D D(66) 0.23 1/0 4.96 Table 4. continued S(2) res type subst’s cvg noc/ dist antn N(15) (%) bb (A˚ ) .(12)H .(12)G Q(1)EK 417 Q Q(84) 0.03 77/12 2.65 .(13)RT Table 4. 457 P P(84)R 0.03 37/12 3.58 The top 25% of residues in 3kscE at the interface with 3kscD. (Field names: res: residue number in the PDB entry; type: amino acid type; .(14)S substs: substitutions seen in the alignment; with the percentage of each type 460 V V(83)S 0.03 30/7 3.99 in the bracket; noc/bb: number of contacts with the ligand, with the number of .(14) contacts realized through backbone atoms given in the bracket; dist: distance L(1)G of closest apporach to the ligand. ) 380 N N(25) 0.04 21/4 3.40 H(60) .(12)KS Table 5. R res type disruptive 395 V V(82)L 0.04 31/1 3.39 mutations .(12) 373 P (Y)(R)(H)(T) A(2)IRC 416 P (YR)(TH)(SCG)(KE) 437 T T(82) 0.04 16/7 2.56 10 C (K)(E)(R)(Q) .(13) 399 G (FKEWR)(H)(MD)(Q) N(2)SP 375 Y (K)(Q)(M)(E) 448 G H(3) 0.05 1/1 4.48 417 Q (Y)(FW)(H)(T) G(77) 457 P (Y)(R)(TH)(ECG) D(3) 460 V (R)(K)(E)(Y) Q(1) 380 N (Y)(T)(FW)(EVCAG) .(14)I 395 V (Y)(E)(R)(K) 451 S Q(3) 0.05 1/1 4.86 437 T (R)(K)(H)(FW) S(77) 448 G (R)(KE)(FWH)(M) .(18)RV 451 S (KR)(FWH)(Y)(M) 393 Q R(7) 0.06 66/8 2.75 continued in next column continued in next column

4 Table 5. continued type in the bracket; noc/bb: number of contacts with the ligand, with the num- res type disruptive ber of contacts realized through backbone atoms given in the bracket; dist: mutations distance of closest apporach to the ligand. ) 393 Q (Y)(T)(FWH)(VCAG) 43 C (E)(KR)(QD)(M) 378 N (Y)(FWH)(TR)(EVCAG) Table 7. 379 A (R)(K)(E)(Y) res type disruptive 476 K (Y)(FW)(T)(VCAG) mutations 394 V (Y)(R)(E)(K) 388 G (E)(KD)(R)(FQMWH) 404 D (FWR)(H)(Y)(VCAG) Table 7. List of disruptive mutations for the top 25% of residues in 3kscE, Table 5. List of disruptive mutations for the top 25% of residues in 3kscE, that are at the interface with glycerol. that are at the interface with 3kscD.

Fig. 7. Residues in 3kscE, at the interface with glycerol, colored by their relative importance. The ligand (glycerol) is colored green. Atoms further Fig. 6. Residues in 3kscE, at the interface with 3kscD, colored by their rela- than 30A˚ away from the geometric center of the ligand, as well as on the line tive importance. 3kscD is shown in backbone representation (See Appendix of sight to the ligand were removed. (See Appendix for the coloring scheme for the coloring scheme for the protein chain 3kscE.) for the protein chain 3kscE.)

Figure 6 shows residues in 3kscE colored by their importance, at the Figure 7 shows residues in 3kscE colored by their importance, at the interface with 3kscD. interface with 3kscBGOL505. Glycerol binding site. By analogy with 3kscB – 3kscBGOL505 Glycerol binding site. By analogy with 3kscB – 3kscBGOL506 interface. Table 6 lists the top 25% of residues at the interface with interface. Table 8 lists the top 25% of residues at the interface with 3kscBGOL505 (glycerol). The following table (Table 7) suggests 3kscBGOL506 (glycerol). The following table (Table 9) suggests possible disruptive replacements for these residues (see Section 3.6). possible disruptive replacements for these residues (see Section 3.6).

Table 6. Table 8. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) 388 G G(86) 0.02 7/7 4.06 50 R R(75) 0.09 6/6 3.92 .(12)RA .(19) K(3)DQ Table 6. The top 25% of residues in 3kscE at the interface with glyce- rol.(Field names: res: residue number in the PDB entry; type: amino acid Table 8. The top 25% of residues in 3kscE at the interface with glyce- type; substs: substitutions seen in the alignment; with the percentage of each rol.(Field names: res: residue number in the PDB entry; type: amino acid

5 type; substs: substitutions seen in the alignment; with the percentage of each Table 10. continued type in the bracket; noc/bb: number of contacts with the ligand, with the num- res type subst’s cvg noc/ dist antn ber of contacts realized through backbone atoms given in the bracket; dist: (%) bb (A˚ ) distance of closest apporach to the ligand. ) Y(1)FKD R 118 D D(81) 0.12 1/0 4.96 Table 9. .(16)I res type disruptive H(1) mutations 50 R (T)(Y)(VCAG)(SD) Table 10. The top 25% of residues in 3kscE at the interface with glyce- rol.(Field names: res: residue number in the PDB entry; type: amino acid type; Table 9. List of disruptive mutations for the top 25% of residues in 3kscE, substs: substitutions seen in the alignment; with the percentage of each type that are at the interface with glycerol. 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 11. res type disruptive mutations 325 N (Y)(T)(FW)(H) 118 D (R)(FCWHG)(KYVA)(T)

Table 11. List of disruptive mutations for the top 25% of residues in 3kscE, that are at the interface with glycerol.

Fig. 8. Residues in 3kscE, at the interface with glycerol, colored by their relative importance. The ligand (glycerol) 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 3kscE.)

Figure 8 shows residues in 3kscE colored by their importance, at the interface with 3kscBGOL506. Glycerol binding site. Table 10 lists the top 25% of residues at the interface with 3kscEGOL499 (glycerol). The following table (Table 11) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 10. Fig. 9. Residues in 3kscE, at the interface with glycerol, colored by their res type subst’s cvg noc/ dist antn relative importance. The ligand (glycerol) is colored green. Atoms further (%) bb (A˚ ) than 30A˚ away from the geometric center of the ligand, as well as on the line 325 N N(79) 0.08 10/6 3.06 site of sight to the ligand were removed. (See Appendix for the coloring scheme S(2) for the protein chain 3kscE.) .(13) continued in next column Figure 9 shows residues in 3kscE colored by their importance, at the interface with 3kscEGOL499.

6 Sulfate ion binding site. Table 12 lists the top 25% of resi- dues at the interface with 3kscBSO4499 (sulfate ion). The following table (Table 13) suggests possible disruptive replacements for these residues (see Section 3.6). Table 12. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 340 G G(85) 0.01 7/7 4.31 .(13)EW 337 P P(68) 0.08 1/1 4.79 R(11)S .(13) E(2)TAQ LK 341 R S(7) 0.14 17/1 2.96 site R(68) .(13) Y(4) V(1)LIF KHTC

Table 12. The top 25% of residues in 3kscE at the interface with sulfate Fig. 10. Residues in 3kscE, at the interface with sulfate ion, colored by their ion.(Field names: res: residue number in the PDB entry; type: amino acid relative importance. The ligand (sulfate ion) is colored green. Atoms further type; substs: substitutions seen in the alignment; with the percentage of each than 30A˚ away from the geometric center of the ligand, as well as on the line type in the bracket; noc/bb: number of contacts with the ligand, with the num- of sight to the ligand were removed. (See Appendix for the coloring scheme ber of contacts realized through backbone atoms given in the bracket; dist: for the protein chain 3kscE.) distance of closest apporach to the ligand. )

Table 14. continued Table 13. res type subst’s cvg noc/ dist antn res type disruptive (%) bb (A˚ ) mutations Y(4) 340 G (KR)(E)(QH)(FMWD) V(1)LIF 337 P (Y)(R)(H)(T) KHTC 341 R (D)(E)(T)(Y) 326 I I(61) 0.23 2/2 4.56 A(7) Table 13. List of disruptive mutations for the top 25% of residues in L(10) 3kscE, that are at the interface with sulfate ion. .(13) V(5)MFT Figure 10 shows residues in 3kscE colored by their importance, at the interface with 3kscBSO4499. Table 14. The top 25% of residues in 3kscE at the interface with glyce- Glycerol binding site. By analogy with 3kscB – 3kscBGOL504 rol.(Field names: res: residue number in the PDB entry; type: amino acid type; interface. Table 14 lists the top 25% of residues at the interface with substs: substitutions seen in the alignment; with the percentage of each type 3kscBGOL504 (glycerol). The following table (Table 15) suggests in the bracket; noc/bb: number of contacts with the ligand, with the number of possible disruptive replacements for these residues (see Section 3.6). contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 14. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Table 15. 363 G V(30) 0.13 2/2 4.88 res type disruptive G(50)L mutations .(13)P 363 G (R)(E)(K)(H) A(2)NI 341 R (D)(E)(T)(Y) 341 R S(7) 0.14 13/0 3.37 site 326 I (R)(Y)(H)(TK) R(68) .(13) Table 15. List of disruptive mutations for the top 25% of residues in continued in next column 3kscE, that are at the interface with glycerol.

7 Table 16. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 152 Q Q(79) 0.14 106/31 2.62 .(15)S P(1)YRL 160 F F(81) 0.14 65/4 3.35 .(15) S(1)AG 125 G G(81) 0.15 31/31 2.69 .(16)YE S(1) 153 L L(79) 0.15 47/13 3.62 .(15)N G(1) V(1)IK 84 P P(75) 0.17 19/1 3.28 .(21)V S(1) L(1)T 151 N N(80) 0.17 11/10 3.81 .(15)T Fig. 11. Residues in 3kscE, at the interface with glycerol, colored by their S(1)AKI relative importance. The ligand (glycerol) is colored green. Atoms further DH than 30A˚ away from the geometric center of the ligand, as well as on the line 108 H H(70) 0.21 50/13 3.53 of sight to the ligand were removed. (See Appendix for the coloring scheme S(5)C for the protein chain 3kscE.) .(16)DI R(1)Q N(1)YA Figure 11 shows residues in 3kscE colored by their importance, at the 65 N P(9) 0.22 26/15 2.96 interface with 3kscBGOL504. N(59) Interface with 3kscF.Table 16 lists the top 25% of residues at the .(19)QG interface with 3kscF. The following table (Table 17) suggests possible S(8)HCT disruptive replacements for these residues (see Section 3.6). M Table 16. 162 L L(78) 0.22 15/7 3.65 res type subst’s cvg noc/ dist antn V(1) (%) bb (A˚ ) .(15)F 86 C C(78) 0.06 1/1 4.39 S-S I(1)WY .(21) 154 D D(69) 0.23 62/10 3.34 129 W W(84) 0.06 29/0 3.63 .(15) .(15)G E(10) 61 P P(77) 0.08 15/2 3.22 R(1)IPG Q(1) MQN .(20)TH 60 R L(57) 0.25 25/1 2.79 58 L L(75) 0.12 15/0 3.82 R(11) .(19)M .(20)Q I(1)S V(6) F(1)Y I(1)FCM 164 G G(82) 0.12 1/1 4.79 .(16)SP Table 16. The top 25% of residues in 3kscE at the interface with 3kscF. A (Field names: res: residue number in the PDB entry; type: amino acid type; 109 Q Q(77) 0.14 38/14 2.98 substs: substitutions seen in the alignment; with the percentage of each type .(16) in the bracket; noc/bb: number of contacts with the ligand, with the number of H(1)KR contacts realized through backbone atoms given in the bracket; dist: distance E(2)N of closest apporach to the ligand. ) continued in next column

8 Table 17. res type disruptive mutations 86 C (KER)(FQMWHD)(NLPI)(Y) 129 W (KE)(D)(Q)(TNR) 61 P (R)(Y)(TH)(E) 58 L (R)(Y)(TH)(K) 164 G (R)(K)(E)(H) 109 Q (Y)(T)(FW)(VCAHG) 152 Q (Y)(H)(T)(FW) 160 F (K)(E)(Q)(D) 125 G (KR)(Q)(H)(FEMW) 153 L (Y)(R)(H)(T) 84 P (R)(Y)(H)(K) 151 N (Y)(FWH)(TR)(E) 108 H (E)(TD)(M)(Q) 65 N (Y)(H)(FW)(R) 162 L (R)(Y)(K)(T) 154 D (R)(H)(FW)(Y) 60 R (T)(D)(Y)(E)

Table 17. List of disruptive mutations for the top 25% of residues in 3kscE, that are at the interface with 3kscF.

Fig. 12. Residues in 3kscE, at the interface with 3kscF, colored by their rela- tive importance. 3kscF is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 3kscE.)

Figure 12 shows residues in 3kscE colored by their importance, at the interface with 3kscF. Glycerol binding site. By analogy with 3kscC – 3kscCGOL500 interface. Table 18 lists the top 25% of residues at the interface with 3kscCGOL500 (glycerol). The following table (Table 19) suggests possible disruptive replacements for these residues (see Section 3.6).

9 Table 18. Table 19. continued res type subst’s cvg noc/ dist res type disruptive (%) bb (A˚ ) mutations 436 K K(68) 0.16 13/0 2.37 376 N (Y)(FWH)(E)(T) T(7) L(1) Table 19. List of disruptive mutations for the top 25% of residues in .(13) 3kscE, that are at the interface with glycerol. E(1) N(2)IFQ R(1)V 432 Y Y(38) 0.19 9/0 3.08 W(44) .(14) F(1)CVL R 374 H H(48) 0.20 17/0 2.93 S(8) F(11) .(12) Q(6) Y(3) D(1) K(3) R(2)PVA TL 381 S S(58) 0.20 6/0 3.14 A(11) R(2) .(11)I E(3) G(2) T(3) N(2)QKC Fig. 13. Residues in 3kscE, at the interface with glycerol, colored by their 376 N N(64) 0.21 11/0 2.54 relative importance. The ligand (glycerol) is colored green. Atoms further .(17) than 30A˚ away from the geometric center of the ligand, as well as on the line V(1) of sight to the ligand were removed. (See Appendix for the coloring scheme T(5) for the protein chain 3kscE.) R(2) H(2)CA Figure 13 shows residues in 3kscE colored by their importance, at the K(1)P interface with 3kscCGOL500. S(2) Glycerol binding site. By analogy with 3kscC – 3kscCGOL499 interface. Table 20 lists the top 25% of residues at the interface with Table 18. The top 25% of residues in 3kscE at the interface with glyce- 3kscCGOL499 (glycerol). The following table (Table 21) suggests rol.(Field names: res: residue number in the PDB entry; type: amino acid type; possible disruptive replacements for these residues (see Section 3.6). substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of Table 20. contacts realized through backbone atoms given in the bracket; dist: distance res type subst’s cvg noc/ dist of closest apporach to the ligand. ) (%) bb (A˚ ) 351 P P(75) 0.11 14/4 3.54 Table 19. H(3) Q(1) res type disruptive .(13) mutations L(3)RAN 436 K (Y)(T)(FW)(CG) 432 Y (K)(Q)(E)(M) S(1) 374 H (E)(D)(T)(Q) 381 S (R)(FWH)(K)(Y) Table 20. The top 25% of residues in 3kscE at the interface with glyce- continued in next column rol.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the number of

10 contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 21. res type disruptive mutations 351 P (Y)(R)(T)(H)

Table 21. List of disruptive mutations for the top 25% of residues in 3kscE, that are at the interface with glycerol.

Fig. 15. A possible active surface on the chain 3kscE. The larger cluster it belongs to is shown in blue.

Table 22. continued res type substitutions(%) cvg H(1)E(1) 50 R R(75).(19)K(3)D 0.09 Q 20 P P(75)F.(19)VHA 0.10 S(1) 411 R Q(72)R(4).(13) 0.15 H(4)D(1)SE(1)G 32 E E(76)Q(2).(19)D 0.16 A

Table 22. Residues forming surface ”patch” in 3kscE.

Table 23. res type disruptive mutations 410 G (R)(FKEW)(H)(M) 50 R (T)(Y)(VCAG)(SD) 20 P (R)(Y)(K)(E) Fig. 14. Residues in 3kscE, at the interface with glycerol, colored by their 411 R (T)(YD)(VA)(CLPIG) relative importance. The ligand (glycerol) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line 32 E (H)(FW)(Y)(R) of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 3kscE.) Table 23. Disruptive mutations for the surface patch in 3kscE.

Figure 14 shows residues in 3kscE colored by their importance, at the Another group of surface residues is shown in Fig.16. The right panel interface with 3kscCGOL499. shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 24, 2.4.3 Possible novel functional surfaces at 25% coverage. One while Table 25 suggests possible disruptive replacements for these group of residues is conserved on the 3kscE surface, away from (or residues (see Section 3.6). susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 3ksc. It is shown in Fig. 15. The right panel Table 24. shows (in blue) the rest of the larger cluster this surface belongs to. res type substitutions(%) cvg antn The residues belonging to this surface ”patch” are listed in Table 22, 340 G G(85).(13)EW 0.01 while Table 23 suggests possible disruptive replacements for these 333 D D(83).(13)E(1)Y 0.04 residues (see Section 3.6). H 337 P P(68)R(11)S 0.08 Table 22. .(13)E(2)TAQLK res type substitutions(%) cvg 341 R S(7)R(68).(13) 0.14 site 410 G G(82).(13)Q(1) 0.05 Y(4)V(1)LIFKHTC continued in next column continued in next column

11 Fig. 16. Another possible active surface on the chain 3kscE. The larger cluster it belongs to is shown in blue.

Table 24. continued res type substitutions(%) cvg antn 332 P A(66)GP(13) 0.18 .(13)S(3)T(1)FR

Table 24. Residues forming surface ”patch” in 3kscE.

Fig. 17. Another possible active surface on the chain 3kscE. Table 25. res type disruptive Table 26. continued mutations res type substitutions(%) cvg antn 340 G (KR)(E)(QH)(FMWD) 129 W W(84).(15)G 0.06 333 D (R)(FVAW)(KCG)(H) 393 Q R(7)Q(75)Y.(12) 0.06 337 P (Y)(R)(H)(T) E(3)P 341 R (D)(E)(T)(Y) 378 N N(79)A(2).(12) 0.07 332 P (R)(Y)(H)(KE) S(1)D(1)KTQ 61 P P(77)Q(1).(20)T 0.08 Table 25. Disruptive mutations for the surface patch in 3kscE. H 359 S S(71)A(6).(13) 0.08 Another group of surface residues is shown in Fig.17. The residues G(4)QNT(1)DCR belonging to this surface ”patch” are listed in Table 26, while Table 360 A A(77).(13)SV(6) 0.09 27 suggests possible disruptive replacements for these residues (see I(1)FD Section 3.6). 379 A A(77)C(2)S(5) 0.09 .(12)G(1)D Table 26. 45 G G(76).(19)R(1) 0.10 res type substitutions(%) cvg antn N(1)WS 373 P P(87).(12)A 0.00 402 V V(79).(12)I(2)A 0.10 318 C C(83).(16) 0.01 S-S L(1)FMSR 416 P P(86).(13) 0.01 80 G G(76).(21)SEQ 0.11 399 G G(85).(12)ERC 0.02 351 P P(75)H(3)Q(1) 0.11 375 Y W(67)F(1)Y(17) 0.03 .(13)L(3)RAN .(12)G S(1) 417 Q Q(84).(13)RT 0.03 58 L L(75).(19)MI(1) 0.12 380 N N(25)H(60).(12) 0.04 SF(1)Y KSR 145 D D(80).(15)H(2)E 0.12 395 V V(82)L.(12)A(2) 0.04 NG IRC 109 Q Q(77).(16)H(1)K 0.14 437 T T(82).(13)N(2)S 0.04 RE(2)N P 152 Q Q(79).(15)SP(1) 0.14 86 C C(78).(21) 0.06 S-S YRL continued in next column continued in next column

12 Table 26. continued Table 27. res type substitutions(%) cvg antn res type disruptive 160 F F(81).(15)S(1)A 0.14 mutations G 373 P (Y)(R)(H)(T) 125 G G(81).(16)YE 0.15 318 C (KER)(FQMWHD)(NLPI)(Y) S(1) 416 P (YR)(TH)(SCG)(KE) 153 L L(79).(15)NG(1) 0.15 399 G (FKEWR)(H)(MD)(Q) V(1)IK 375 Y (K)(Q)(M)(E) 403 F F(72).(12)Y(4) 0.15 417 Q (Y)(FW)(H)(T) L(8)M(1)HV 380 N (Y)(T)(FW)(EVCAG) 346 T N(69)T(10).(13) 0.16 395 V (Y)(E)(R)(K) H(1)D(3)YSQ 437 T (R)(K)(H)(FW) 436 K K(68)T(7)L(1) 0.16 86 C (KER)(FQMWHD)(NLPI)(Y) .(13)E(1)N(2)IF 129 W (KE)(D)(Q)(TNR) QR(1)V 393 Q (Y)(T)(FWH)(VCAG) 84 P P(75).(21)VS(1) 0.17 378 N (Y)(FWH)(TR)(EVCAG) L(1)T 61 P (R)(Y)(TH)(E) 85 G G(76).(21)LS(1) 0.17 359 S (R)(K)(FWH)(M) A 360 A (R)(K)(Y)(E) 151 N N(80).(15)TS(1) 0.17 379 A (R)(K)(E)(Y) AKIDH 45 G (E)(K)(R)(D) 15 L I(11)L(66).(20) 0.19 402 V (Y)(E)(R)(K) RHD 80 G (R)(FWH)(K)(E) 131 Y Y(77)D.(15)F(2) 0.20 351 P (Y)(R)(T)(H) H(3)T 58 L (R)(Y)(TH)(K) 374 H H(48)S(8)F(11) 0.20 145 D (R)(FW)(H)(Y) .(12)Q(6)Y(3) 109 Q (Y)(T)(FW)(VCAHG) D(1)K(3)R(2)PVA 152 Q (Y)(H)(T)(FW) TL 160 F (K)(E)(Q)(D) 381 S S(58)A(11)R(2) 0.20 125 G (KR)(Q)(H)(FEMW) .(11)IE(3)G(2) 153 L (Y)(R)(H)(T) T(3)N(2)QKC 403 F (K)(E)(T)(Q) 46 V V(68).(19)LF(4) 0.21 346 T (R)(K)(FMW)(H) ST(5)I(1) 436 K (Y)(T)(FW)(CG) 108 H H(70)S(5)C.(16) 0.21 84 P (R)(Y)(H)(K) DIR(1)QN(1)YA 85 G (R)(K)(E)(H) 376 N N(64).(17)V(1) 0.21 151 N (Y)(FWH)(TR)(E) T(5)R(2)H(2)CA 15 L (Y)(TR)(CHG)(S) K(1)PS(2) 131 Y (K)(Q)(M)(R) 65 N P(9)N(59).(19)Q 0.22 374 H (E)(D)(T)(Q) GS(8)HCTM 381 S (R)(FWH)(K)(Y) 162 L L(78)V(1).(15)F 0.22 46 V (R)(K)(E)(Y) I(1)WY 108 H (E)(TD)(M)(Q) 154 D D(69).(15)E(10) 0.23 376 N (Y)(FWH)(E)(T) R(1)IPGMQN 65 N (Y)(H)(FW)(R) 404 D D(66)S(2)N(15) 0.23 162 L (R)(Y)(K)(T) .(12)HQ(1)EK 154 D (R)(H)(FW)(Y) 110 K K(74).(16)R(5) 0.24 404 D (FWR)(H)(Y)(VCAG) Q(1)NVP 110 K (Y)(T)(FW)(S) 60 R L(57)R(11).(20) 0.25 60 R (T)(D)(Y)(E) QV(6)I(1)FCM 441 A (R)(K)(Y)(E) 441 A A(54)P(19)S(10) 0.25 .(14)TGDR Table 27. Disruptive mutations for the surface patch in 3kscE.

Table 26. Residues forming surface ”patch” in 3kscE.

13 3 NOTES ON USING TRACE RESULTS 3.6 Mutation suggestions 3.1 Coverage Mutation suggestions are completely heuristic and based on comple- Trace results are commonly expressed in terms of coverage: the resi- mentarity with the substitutions found in the alignment. Note that due is important if its “coverage” is small - that is if it belongs to they are meant to be disruptive to the interaction of the protein some small top percentage of residues [100% is all of the residues with its ligand. The attempt is made to complement the following in a chain], according to trace. The ET results are presented in the properties: small [AV GSTC], medium [LPNQDEMIK], large form of a table, usually limited to top 25% percent of residues (or [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to some nearby percentage), sorted by the strength of the presumed tively [KHR], or negatively [DE] charged, aromatic [WFYH], evolutionary pressure. (I.e., the smaller the coverage, the stronger the long aliphatic chain [EKRQM], OH-group possession [SDETY ], pressure on the residue.) Starting from the top of that list, mutating a and NH2 group possession [NQRK]. The suggestions are listed couple of residues should affect the protein somehow, with the exact according to how different they appear to be from the original amino effects to be determined experimentally. acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid 3.2 Known substitutions types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 4 APPENDIX an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, 4.1 File formats one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file: is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough • alignment# number of the position in the alignment guide - due to rounding errors these percentages often do not add up • residue# residue number in the PDB file to 100%. • type amino acid type • 3.3 Surface rank rank of the position according to older version of ET • variability To detect candidates for novel functional interfaces, first we look for has two subfields: residues that are solvent accessible (according to DSSP program) by 1. number of different amino acids appearing in in this column 2 at least 10A˚ , which is roughly the area needed for one water mole- of the alignment cule to come in the contact with the residue. Furthermore, we require 2. their type that these residues form a “cluster” of residues which have neighbor • rho ET score - the smaller this value, the lesser variability of within 5A˚ from any of their heavy atoms. this position across the branches of the tree (and, presumably, Note, however, that, if our picture of protein evolution is correct, the greater the importance for the protein) 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- • sing the set for mutagenesis. (Especially if they form a cluster with gaps percentage of gaps in this column the surface residues.) 4.2 Color schemes used 3.4 Number of contacts The following color scheme is used in figures with residues colored 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, mutation presumably won’t have strong impact). Two heavy atoms DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, A˚ are considered to be “in contact” if their centers are closer than 5 . tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 3.5 Annotation evolutionary pressure they experience can be seen in Fig. 18. If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” 4.3 Credits appears. Annotations carried over from PDB are the following: site 4.3.1 Alistat alistat reads a multiple sequence alignment from the (indicating existence of related site record in PDB ), S-S (disulfide file and shows a number of simple statistics about it. These stati- bond forming residue), hb (hydrogen bond forming residue, jb (james stics include the format, the number of sequences, the total number bond forming residue), and sb (for salt bridge forming residue). of residues, the average and range of the sequence lengths, and the

14 ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. http://www.drive5.com/muscle/

COVERAGE 4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol V is an open-source application copyrighted by DeLano Scien- 100% 50% 30% 5% 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 the scripts and launch the viewer.) 4.4 Note about ET Viewer V Dan Morgan from the Lichtarge lab has developed a visualization RELATIVE IMPORTANCE tool specifically for viewing trace results. If you are interested, please visit:

Fig. 18. Coloring scheme used to color residues by their relative importance. http://mammoth.bcm.tmc.edu/traceview/ The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. 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- 2 is copyrighted by Lichtarge Lab, Baylor College of Medicine, 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, • 3kscE.complex.pdb - coordinates of 3kscE 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- • 3kscE.etvx - ET viewer input file for 3kscE gnment as a starting point for the analysis (sequences shorter than • 3kscE.cluster report.summary - Cluster report summary for 75% of the query are taken out, however); R. Schneider, A. de 3kscE Daruvar, and C. Sander. ”The HSSP database of protein structure- • 3kscE.ranks - Ranks file in sequence order for 3kscE sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 3kscE.clusters - Cluster descriptions for 3kscE http://swift.cmbi.kun.nl/swift/hssp/ • 3kscE.msf - the multiple sequence alignment used for the chain 3kscE 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 3kscE.descr - description of sequences used in 3kscE msf Wesley,” Reading, Mass. (1986). • 3kscE.ranks sorted - full listing of residues and their ranking for 4.3.6 Muscle When making alignments “from scratch”, report 3kscE maker uses Muscle alignment program: Edgar, Robert C. (2004), • 3kscE.3kscB.if.pml - Pymol script for Figure 5

15 • 3kscE.cbcvg - used by other 3kscE – related pymol scripts • 3kscE.3kscBGOL504.if.pml - Pymol script for Figure 11 • 3kscE.3kscD.if.pml - Pymol script for Figure 6 • 3kscE.3kscF.if.pml - Pymol script for Figure 12 • 3kscE.3kscBGOL505.if.pml - Pymol script for Figure 7 • 3kscE.3kscCGOL500.if.pml - Pymol script for Figure 13 • 3kscE.3kscBGOL506.if.pml - Pymol script for Figure 8 • 3kscE.3kscCGOL499.if.pml - Pymol script for Figure 14 • 3kscE.3kscEGOL499.if.pml - Pymol script for Figure 9 • 3kscE.3kscBSO4499.if.pml - Pymol script for Figure 10

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