Pages 1–9 1zyz Evolutionary trace report by report maker September 8, 2008

4.3.1 Alistat 8 4.3.2 CE 8 4.3.3 DSSP 8 4.3.4 HSSP 8 4.3.5 LaTex 8 4.3.6 Muscle 8 4.3.7 Pymol 8 4.4 Note about ET Viewer 8 4.5 Citing this work 9 4.6 About report maker 9 4.7 Attachments 9

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1zyz): Title: Structures of yeast ribonucloetide reductase i Compound: Mol id: 1; molecule: ribonucleoside-diphosphate reduc- tase large chain 1; chain: a, b; synonym: ribonucleotide reductase i, rnr1; ec: 1.17.4.1; engineered: yes Organism, scientific name: Saccharomyces Cerevisiae; 1zyz contains a single unique chain 1zyzA (776 residues long) and its homologue 1zyzB. CONTENTS 2 CHAIN 1ZYZA 1 Introduction 1 2.1 P21524 overview 2 Chain 1zyzA 1 From SwissProt, id P21524, 97% identical to 1zyzA: 2.1 P21524 overview 1 Description: Ribonucleoside-diphosphate reductase large chain 1 2.2 Multiple sequence alignment for 1zyzA 1 (EC 1.17.4.1) (Ribonucleotide reductase). 2.3 Residue ranking in 1zyzA 1 Organism, scientific name: Saccharomyces cerevisiae (Baker’s 2.4 Top ranking residues in 1zyzA and their position on yeast). the structure 1 Taxonomy: Eukaryota; Fungi; Ascomycota; Saccharomycotina; 2.4.1 Clustering of residues at 25% coverage. 2 Saccharomycetes; Saccharomycetales; Saccharomycetaceae; Sac- 2.4.2 Overlap with known functional surfaces at charomyces. 25% coverage. 3 Function: Provides the precursors necessary for DNA synthesis. 2.4.3 Possible novel functional surfaces at 25% Catalyzes the biosynthesis of deoxyribonucleotides from the corre- coverage. 3 sponding ribonucleotides. Catalytic activity: 2’-deoxyribonucleoside diphosphate + thioredo- 3 Notes on using trace results 7 xin disulfide + H(2)O = ribonucleoside diphosphate + thioredoxin. 3.1 Coverage 7 Pathway: DNA replication pathway; first step. 3.2 Known substitutions 7 Subunit: Requires two subunits for activity. Yeast has two large sub- 3.3 Surface 7 units: RNR1 and RNR3. RNR1 is essential for mitotic viability; 3.4 Number of contacts 7 RNR3 is not essential. In damaged cells or cells arrested for DNA 3.5 Annotation 7 synthesis, the reductase consists of multiple species because of the 3.6 Mutation suggestions 7 association of the small subunit (RNR2) with either the constitutively expressed (RNR1) or the inducible (RNR3) large subunits. 4 Appendix 8 Similarity: Belongs to the ribonucleoside diphosphate reductase 4.1 File formats 8 large chain family. 4.2 Color schemes used 8 About: This Swiss-Prot entry is copyright. It is produced through a 4.3 Credits 8 collaboration between the Swiss Institute of Bioinformatics and the

1 Lichtarge lab 2006 Fig. 1. Residues 15-278 in 1zyzA colored by their relative importance. (See Fig. 2. Residues 279-536 in 1zyzA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.) Appendix, Fig.8, for the coloring scheme.)

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2.2 Multiple sequence alignment for 1zyzA For the chain 1zyzA, the alignment 1zyzA.msf (attached) with 106 sequences was used. The alignment was assembled through combi- nation of BLAST searching on the UniProt database and alignment using Muscle program. It can be found in the attachment to this report, under the name of 1zyzA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 106 Fig. 3. Residues 537-796 in 1zyzA colored by their relative importance. (See Total number of residues: 77219 Appendix, Fig.8, for the coloring scheme.) Smallest: 583 Largest: 776 Average length: 728.5 2.4 Top ranking residues in 1zyzA and their position on Alignment length: 776 the structure Average identity: 38% In the following we consider residues ranking among top 25% of Most related pair: 99% residues in the protein . Figure 4 shows residues in 1zyzA colored Most unrelated pair: 18% by their importance: bright red and yellow indicate more conser- Most distant seq: 45% ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 33% eukaryotic ( 6% vertebrata, 8% 2.4.1 Clustering of residues at 25% coverage. Fig. 5 shows the fungi, 8% plantae), 40% prokaryotic, and 25% viral sequences. (Des- top 25% of all residues, this time colored according to clusters they criptions of some sequences were not readily available.) The file belong to. The clusters in Fig.5 are composed of the residues listed containing the sequence descriptions can be found in the attachment, in Table 1. under the name 1zyzA.descr. Table 1. cluster size member 2.3 Residue ranking in 1zyzA color residues The 1zyzA sequence is shown in Figs. 1–3, with each residue colored red 168 147,150,151,154,155,156,165 according to its estimated importance. The full listing of residues 173,199,200,201,202,203,204 in 1zyzA can be found in the file called 1zyzA.ranks sorted in the continued in next column attachment.

2 Table 1. continued cluster size member color residues 262,271,274,275,277,280,283 287,288,290,293,294,295,296 298,300,302,304,305,307,311 314,315,319,324,326,329,331 333,335,336,337,338,339,341 342,348,350,352,353,359,364 367,371,374,375,385,389,396 399,400,402,403,407,408,411 422,425,426,427,428,430,431 433,441,442,443,444,445,447 449,450,481,482,485,488,503 504,506,508,510,511,513,514 528,531,532,543,547,551,555 557,563,568,571,573,596,598 599,600,604,605,606,607,608 609,610,611,612,613,614,616 618,619,622,623,688,698,699 702,707,708,710,711,713,728 732,735,737,738,741,742,744 Fig. 4. Residues in 1zyzA, colored by their relative importance. Clockwise: blue 7 631,632,633,636,637,638,640 front, back, top and bottom views. yellow 6 627,642,645,649,668,684 green 3 168,190,194 purple 3 537,585,588 azure 2 228,231

Table 1. Clusters of top ranking residues in 1zyzA.

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. Interface with 1zyzB.Table 2 lists the top 25% of residues at the interface with 1zyzB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 1 16/16 0.00 2 48/48 0.00 744 R R(75) 0.09 5/4 3.72 K(11) H(3) N(8)Q Fig. 5. Residues in 1zyzA, colored according to the cluster they belong to: 555 G G(90) 0.14 13/13 3.34 red, followed by blue and yellow are the largest clusters (see Appendix for L(4) the coloring scheme). Clockwise: front, back, top and bottom views. The A(2) corresponding Pymol script is attached. C(1) 551 A A(80) 0.20 6/6 3.35 Table 1. continued C(14) cluster size member V(1)GL color residues S(1) 205,207,209,210,214,215,216 217,218,219,220,239,241,242 Table 2. The top 25% of residues in 1zyzA at the interface with 1zyzB. 243,245,246,247,248,249,256 (Field names: res: residue number in the PDB entry; type: amino acid type; continued in next column substs: substitutions seen in the alignment; with the percentage of each type

3 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 3. res type disruptive mutations 1 2 744 R (T)(YD)(VCAG)(S) 555 G (R)(KE)(H)(QD) 551 A (R)(K)(E)(Y) Fig. 7. A possible active surface on the chain 1zyzA. The larger cluster it belongs to is shown in blue. Table 3. List of disruptive mutations for the top 25% of residues in 1zyzA, that are at the interface with 1zyzB. Table 4. res type substitutions(%) cvg 155 Y Y(100) 0.02 165 E E(100) 0.02 218 C C(100) 0.02 247 G G(100) 0.02 426 N N(100) 0.02 427 L L(100) 0.02 428 C C(100) 0.02 430 E E(100) 0.02 445 L L(100) 0.02 503 R R(100) 0.02 607 P P(100) 0.02 735 G G(100) 0.02 203 P P(96)S(3) 0.03 217 S S(96)N(3) 0.03 293 R R(96)K(3) 0.03 350 L L(99)F 0.03 400 G G(96)S(3) 0.03 324 R R(92)K(7) 0.04 737 K K(99)A 0.04 741 Y Y(99)L 0.04 742 Y Y(99)R 0.04 246 G G(87)S(3)A(8) 0.05 295 G G(83)T(12)V(3) 0.05 608 T T(86)S(8)N(4) 0.05 Fig. 6. Residues in 1zyzA, at the interface with 1zyzB, colored by their rela- 623 P P(98)A(1) 0.05 tive importance. 1zyzB is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1zyzA.) 702 R R(87)A(3)M(8) 0.05 307 D D(82)Q(3)E(14) 0.06 447 S S(86)N(4)A(8) 0.06 Figure 6 shows residues in 1zyzA colored by their importance, at the 568 G G(99)N 0.06 interface with 1zyzB. 636 G G(91)E(3)F(4) 0.06 209 G G(85)R(12)A(1) 0.07 2.4.3 Possible novel functional surfaces at 25% coverage. One 598 R R(86)Y(9)F(3) 0.07 group of residues is conserved on the 1zyzA surface, away from (or 606 M M(67)A(32) 0.07 susbtantially larger than) other functional sites and interfaces reco- 710 H Q(81)I(16)H(1) 0.07 gnizable in PDB entry 1zyz. It is shown in Fig. 7. The right panel 738 T T(89)S(9)Q 0.07 shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table continued in next column 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6).

4 Table 4. continued Table 4. continued res type substitutions(%) cvg res type substitutions(%) cvg 205 L L(77)M(22) 0.08 S(1)D 642 N N(82)V(12)A(4)H 0.08 290 G G(78)E(5)N(1) 0.15 707 D D(92)C(7) 0.08 S(13)A 173 R R(83)A(3)L(8) 0.09 326 R R(54)H(24)D(12) 0.15 T(3)V L(5)N(1)K 611 T T(51)I(27)S(20) 0.09 262 I I(79).(14)V(5)A 0.16 732 W W(75)Y(21)F(2) 0.09 294 P P(38)K(43)F(2) 0.16 744 R R(75)K(11)H(3) 0.09 R(5)LG(8) N(8)Q 296 A A(67)G(14)V(2) 0.16 207 N N(74)F(9)S(5) 0.10 S(15) G(8)A(1) 514 D D(63)Q(3)T(12) 0.16 271 G G(93)N(1)S(2) 0.10 N(1)S(5)Y(8) .(1) H(4) 557 Y Y(64)F(16)C(14) 0.10 199 T T(47)M(20)V(11) 0.17 H(2)L(2) C(3)L(6)S(8) 713 N N(84)T(13)S(1) 0.10 I(1) 202 S S(34)T(61)G(3) 0.11 214 Q RQ(79)S(8)L(5) 0.17 353 P P(87)S(5)R(2) 0.11 Y(2)N(1)A D(2)H 231 I I(83)T(11)A(3)L 0.17 287 D D(49)N(33)S(17) 0.12 R 329 F F(66)N(16)H(5) 0.12 319 G G(87)A(3)P(3) 0.17 D(9)L(1) R(1)T(1)L 342 V V(73)Y(12)M 0.12 482 N N(55)D(38)T(2) 0.17 L(12)A Q(2) 618 N N(43)T(24)S(28) 0.12 573 D D(83)Q(1).(5) 0.17 Y(3) E(2)R(3)A(1)K 728 H H(50)Y(21)L(27) 0.12 150 T T(69)S(14)V(1) 0.18 Q Q(8)L(3)P(1) 304 W W(92)GH(2)F(3) 0.13 315 R K(34)R(65) 0.18 375 E E(82)V(10).(5)I 0.13 610 S S(62)A(8)G(3) 0.18 A T(25) 399 T T(95)S(2)Q(1) 0.13 151 L L(84)F(3)Y(6) 0.19 513 A A(59)H(21)Q(14) 0.13 M(2)V(1) T(3)N 156 L L(80)V(3)F(13) 0.19 609 A A(80)S(4)V(2) 0.13 G(2) E(8)M(3) 280 N N(65)D(25)SQ(8) 0.19 614 I I(82)L(7)V(10) 0.13 616 G G(75)E(9)S(1) 0.19 201 A A(66)S(22)P(10) 0.14 D(2)N(10) 336 D D(87)E(1)R(1) 0.14 637 E E(70)M(3)K(4) 0.19 S(1)K(6) S(4)I(8)R(3) 374 Y Y(68)FL(16).(5) 0.14 D(2)A A(3)V(3)C 239 A A(69)V(19)S(4) 0.20 555 G G(90)L(4)A(2) 0.14 M(3)I(1) C(1) 288 Q Q(80)V(3)D(3) 0.20 613 Q Q(71)I(9)N(10) 0.14 R(3)K(2)S(2) L(3)S(3)M A(1)G 631 R R(50)E(8)K(34) 0.14 341 R R(84)KL(1)A(8) 0.20 H(4)Q M(1)QC 688 W W(55)Y(10)F(33) 0.14 551 A A(80)C(14)V(1)G 0.20 243 K K(69)L(7)A(7) 0.15 LS(1) S(13)N(1) 571 Q Q(44)P(28)H(16) 0.20 256 R R(83)N(10).(2) 0.15 .(9)D continued in next column continued in next column

5 Table 4. continued Table 5. res type substitutions(%) cvg res type disruptive 633 V V(42)N(14)T(26) 0.20 mutations S(12)K(4) 155 Y (K)(QM)(NEVLAPIR)(D) 210 T T(81)R(8)L(1)A 0.21 165 E (FWH)(YVCARG)(T)(SNKLPI) K(4)V(1)S 218 C (KER)(FQMWHD)(NYLPI)(SVA) 245 A A(53)G(17)R(19) 0.21 247 G (KER)(FQMWHD)(NYLPI)(SVA) K(1)T(3)N(2) 426 N (Y)(FTWH)(SEVCARG)(MD) 337 L L(84)E(9)I(1) 0.21 427 L (YR)(TH)(SKECG)(FQWD) A(1)YH 428 C (KER)(FQMWHD)(NYLPI)(SVA) 488 N N(65)S(8)G(5) 0.21 430 E (FWH)(YVCARG)(T)(SNKLPI) T(6)A(5)Q(8) 445 L (YR)(TH)(SKECG)(FQWD) 275 M M(42)F(35)V(6) 0.22 503 R (TD)(SYEVCLAPIG)(FMW)(N) C(3)A(4)W(5)L 607 P (YR)(TH)(SKECG)(FQWD) 314 I L(75)M(12)A(3) 0.22 735 G (KER)(FQMWHD)(NYLPI)(SVA) I(8) 203 P (R)(Y)(H)(K) 622 E E(75)A(4)Y(4) 0.22 217 S (R)(FKWH)(YM)(EQ) D(8)S(3)L(1)Q 293 R (T)(YD)(SVCAG)(FELWPI) 632 R R(42)Q(3)V(14) 0.22 350 L (R)(TY)(KE)(SCHG) S(14)K(5)TA(8) 400 G (KR)(E)(FQMWH)(D) E(8)LI 324 R (T)(YD)(SVCAG)(FELWPI) 692 Q Q(67)AP(13)T(4) 0.22 737 K (Y)(FTW)(SCHDG)(EVA) N(9)M(2)V 741 Y (K)(QR)(EM)(NVA) 228 I I(47)T(7)E(3) 0.23 742 Y (KM)(EVQLAPI)(ND)(SCRG) L(36)N(2)S(1) 246 G (KR)(E)(QH)(FMW) 352 S C(39)D(29)A(1) 0.23 295 G (KR)(E)(QH)(FMW) S(28)T 608 T (R)(K)(FWH)(M) 619 E E(61)Q(11)A(10) 0.23 623 P (YR)(H)(TKE)(SQCDG) P(6)N(8)D(1) 702 R (TYD)(E)(S)(CG) 698 M M(45)A(14)L(22) 0.23 307 D (FWHR)(Y)(VCAG)(T) R(3)V(3)H(3) 447 S (R)(K)(H)(FW) Q(4)YF 568 G (ER)(FKWH)(YMD)(Q) 154 S S(40)R(32)E(5) 0.24 636 G (K)(R)(E)(QH) T(3)F(3)M(1) 209 G (E)(D)(K)(R) K(8)AG(1)L 598 R (D)(T)(E)(VCLAPIG) 274 P P(85)H(3)S(2) 0.24 606 M (Y)(H)(TR)(SCDG) G(3).(1)RA 710 H (T)(E)(D)(CG) 277 R R(43)K(47)H(4) 0.24 738 T (R)(FWH)(K)(M) V(1)N(1)T 205 L (Y)(R)(TH)(SCG) 638 F F(65)I(9)L(16) 0.24 642 N (Y)(E)(T)(HR) T(3)R(3)YE 707 D (R)(FWH)(K)(Y) 640 V V(58)TR(11)K(4) 0.24 173 R (D)(E)(Y)(T) E(3)I(8)Q(12) 611 T (R)(K)(H)(FQW) 264 G G(77).(12)S(9)D 0.25 732 W (K)(E)(Q)(D) 389 W W(59)F(19)M(10) 0.25 744 R (T)(YD)(VCAG)(S) A(4)Y(3)L(1) 207 N (Y)(E)(HR)(FW) 600 S S(76)G(4)A(15) 0.25 271 G (R)(KE)(FWH)(M) V(1)LC 557 Y (K)(Q)(E)(M) 621 F I(44)F(49)V(2) 0.25 713 N (Y)(FWH)(R)(E) L(2)T 202 S (KR)(FQMWH)(E)(NLPI) 353 P (Y)(R)(T)(H) Table 4. Residues forming surface ”patch” in 1zyzA. 287 D (R)(FWH)(Y)(K) continued in next column

6 Table 5. continued Table 5. continued res type disruptive res type disruptive mutations mutations 329 F (KE)(T)(QR)(D) 632 R (Y)(T)(D)(E) 342 V (R)(K)(E)(Y) 692 Q (Y)(H)(FW)(T) 618 N (FWHR)(Y)(E)(M) 228 I (R)(Y)(H)(TK) 728 H (E)(T)(D)(QM) 352 S (R)(K)(H)(FQW) 304 W (E)(K)(QD)(T) 619 E (H)(FW)(Y)(R) 375 E (H)(R)(Y)(FW) 698 M (Y)(T)(H)(R) 399 T (R)(FWH)(K)(M) 154 S (R)(K)(H)(FW) 513 A (E)(Y)(R)(K) 274 P (Y)(R)(E)(H) 609 A (YR)(K)(H)(E) 277 R (D)(T)(YE)(SVCLAPIG) 614 I (YR)(H)(T)(KE) 638 F (K)(E)(TQ)(DR) 201 A (R)(KY)(E)(H) 640 V (Y)(HR)(E)(K) 336 D (FW)(HR)(Y)(VCAG) 264 G (R)(K)(FWH)(EQM) 374 Y (K)(Q)(R)(E) 389 W (K)(E)(QD)(T) 555 G (R)(KE)(H)(QD) 600 S (R)(K)(H)(Q) 613 Q (Y)(H)(T)(FW) 621 F (K)(E)(R)(Q) 631 R (T)(Y)(VCADG)(S) 688 W (K)(E)(Q)(D) Table 5. Disruptive mutations for the surface patch in 1zyzA. 243 K (Y)(FW)(T)(H) 256 R (T)(Y)(VCAG)(D) 290 G (R)(K)(H)(E) 326 R (T)(Y)(D)(CG) 3 NOTES ON USING TRACE RESULTS 262 I (YR)(H)(TKE)(SQCDG) 294 P (Y)(R)(T)(H) 3.1 Coverage 296 A (KR)(E)(Y)(QH) Trace results are commonly expressed in terms of coverage: the resi- 514 D (R)(FW)(H)(K) due is important if its “coverage” is small - that is if it belongs to 199 T (R)(K)(H)(FW) some small top percentage of residues [100% is all of the residues 214 Q (Y)(H)(FW)(T) in a chain], according to trace. The ET results are presented in the 231 I (Y)(R)(H)(TE) form of a table, usually limited to top 25% percent of residues (or 319 G (E)(R)(K)(H) to some nearby percentage), sorted by the strength of the presumed 482 N (Y)(FWH)(R)(T) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 573 D (FW)(H)(R)(Y) pressure on the residue.) Starting from the top of that list, mutating a 150 T (R)(K)(H)(FW) couple of residues should affect the protein somehow, with the exact 315 R (T)(YD)(SVCAG)(FELWPI) effects to be determined experimentally. 610 S (KR)(QH)(FMW)(E) 151 L (R)(Y)(TK)(E) 3.2 Known substitutions 156 L (R)(Y)(KE)(H) One of the table columns is “substitutions” - other amino acid types 280 N (Y)(FWH)(TR)(VCAG) seen at the same position in the alignment. These amino acid types 616 G (R)(K)(FWH)(YM) may be interchangeable at that position in the protein, so if one wants 637 E (H)(FYW)(R)(CG) to affect the protein by a point mutation, they should be avoided. For 239 A (YR)(K)(E)(H) example if the substitutions are “RVK” and the original protein has 288 Q (Y)(FWH)(T)(CG) an R at that position, it is advisable to try anything, but RVK. Conver- 341 R (Y)(T)(D)(E) sely, when looking for substitutions which will not affect the protein, 551 A (R)(K)(E)(Y) one may try replacing, R with K, or (perhaps more surprisingly), with 571 Q (Y)(T)(FWH)(CG) V. The percentage of times the substitution appears in the alignment 633 V (Y)(R)(E)(K) is given in the immediately following bracket. No percentage is given 210 T (R)(K)(H)(FW) in the cases when it is smaller than 1%. This is meant to be a rough 245 A (Y)(E)(R)(KD) guide - due to rounding errors these percentages often do not add up 337 L (R)(Y)(T)(H) to 100%. 488 N (Y)(H)(FW)(R) 275 M (Y)(R)(TH)(D) 3.3 Surface 314 I (Y)(R)(H)(T) To detect candidates for novel functional interfaces, first we look for 622 E (H)(FWR)(Y)(CG) residues that are solvent accessible (according to DSSP program) by 2 continued in next column at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms.

7 Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with COVERAGE the surface residues.) V 3.4 Number of contacts 100% 50% 30% 5% Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms V are considered to be “in contact” if their centers are closer than 5A˚ . RELATIVE IMPORTANCE 3.5 Annotation

If the residue annotation is available (either from the pdb file or Fig. 8. Coloring scheme used to color residues by their relative importance. from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide • rho ET score - the smaller this value, the lesser variability of bond forming residue), hb (hydrogen bond forming residue, jb (james this position across the branches of the tree (and, presumably, bond forming residue), and sb (for salt bridge forming residue). the greater the importance for the protein) • cvg 3.6 Mutation suggestions coverage - percentage of the residues on the structure which have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • gaps percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GST C], medium [LP NQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [W F Y H], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDET Y ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 8. disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will 4.3 Credits choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4 APPENDIX of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best • type amino acid type relative) for all N sequences, then finding the minimum of these N • rank rank of the position according to older version of ET numbers (hence, the most outlying sequence). alistat is copyrighted • variability has two subfields: by HHMI/Washington University School of Medicine, 1992-2001, 1. number of different amino acids appearing in in this column and freely distributed under the GNU General Public License. of the alignment 4.3.2 CE To map ligand binding sites from different 2. their type source structures, report maker uses the CE program:

8 http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) The viewer is self-unpacking and self-installing. Input files to be used ”Protein structure alignment by incremental combinatorial extension with ETV (extension .etvx) can be found in the attachment to the (CE) of the optimal path . Protein Engineering 11(9) 739-747. main report. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4.5 Citing this work ˚ 2 ble if the DSSP program finds it exposed to water by at least 10A , The method used to rank residues and make predictions in this report which is roughly the area needed for one water molecule to come in can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by Importance” J. Mol. Bio. 336: 1265-82. For the original version @cmbi.kun.nl by Elmar.Krieger November 18,2002, of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- http://www.cmbi.kun.nl/gv/dssp/descrip.html. tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- report maker itself is described in Mihalek I., I. Res and O. gnment as a starting point for the analysis (sequences shorter than Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 75% of the query are taken out, however); R. Schneider, A. de of service for comparative analysis of proteins.” Bioinformatics Daruvar, and C. Sander. ”The HSSP database of protein structure- 22:1656-7. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 4.6 About report maker http://swift.cmbi.kun.nl/swift/hssp/ report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.5 LaTex The text for this report was processed using LATEX; is copyrighted by Lichtarge Lab, Baylor College of Medicine, Leslie Lamport, “LaTeX: A Document Preparation System Addison- Houston. Wesley,” Reading, Mass. (1986). 4.7 Attachments 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), The following files should accompany this report: ”MUSCLE: multiple sequence alignment with high accuracy and • 1zyzA.complex.pdb - coordinates of 1zyzA with all of its high throughput.” Nucleic Acids Research 32(5), 1792-97. interacting partners http://www.drive5.com/muscle/ • 1zyzA.etvx - ET viewer input file for 1zyzA • 4.3.7 Pymol The figures in this report were produced using 1zyzA.cluster report.summary - Cluster report summary for Pymol. The scripts can be found in the attachment. Pymol 1zyzA is an open-source application copyrighted by DeLano Scien- • 1zyzA.ranks - Ranks file in sequence order for 1zyzA tific LLC (2005). For more information about Pymol see • 1zyzA.clusters - Cluster descriptions for 1zyzA http://pymol.sourceforge.net/. (Note for Windows • 1zyzA.msf - the multiple sequence alignment used for the chain users: the attached package needs to be unzipped for Pymol to read 1zyzA the scripts and launch the viewer.) • 1zyzA.descr - description of sequences used in 1zyzA msf 4.4 Note about ET Viewer • 1zyzA.ranks sorted - full listing of residues and their ranking for Dan Morgan from the Lichtarge lab has developed a visualization 1zyzA tool specifically for viewing trace results. If you are interested, please • 1zyzA.1zyzB.if.pml - Pymol script for Figure 6 visit: • 1zyzA.cbcvg - used by other 1zyzA – related pymol scripts http://mammoth.bcm.tmc.edu/traceview/

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