Pages 1–11 1aj8 Evolutionary trace report by report maker November 10, 2009

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

1 INTRODUCTION From the original Data Bank entry (PDB id 1aj8): Title: from pyrococcus furiosus Compound: Mol id: 1; molecule: citrate synthase; chain: a, b; ec: 4.1.3.7; engineered: yes Organism, scientific name: Pyrococcus Furiosus; 1aj8 contains a single unique chain 1aj8A (371 residues long) and its homologue 1aj8B.

CONTENTS 2 CHAIN 1AJ8A 1 Introduction 1 2.1 Q53554 overview 2 Chain 1aj8A 1 From SwissProt, id Q53554, 100% identical to 1aj8A: 2.1 Q53554 overview 1 Description: Citrate synthase (EC 2.3.3.1). 2.2 Multiple sequence alignment for 1aj8A 1 Organism, scientific name: Pyrococcus furiosus. 2.3 Residue ranking in 1aj8A 1 Taxonomy: ; Euryarchaeota; Thermococci; Thermococca- 2.4 Top ranking residues in 1aj8A and their position on les; Thermococcaceae; Pyrococcus. the structure 2 Catalytic activity: Acetyl-CoA + H(2)O + oxaloacetate = citrate + 2.4.1 Clustering of residues at 25% coverage. 2 CoA. 2.4.2 Overlap with known functional surfaces at Pathway: Tricarboxylic acid cycle. 25% coverage. 2 Subunit: Homodimer. Similarity: Belongs to the citrate synthase family. 3 Notes on using trace results 9 About: This Swiss-Prot entry is copyright. It is produced through a 3.1 Coverage 9 collaboration between the Swiss Institute of Bioinformatics and the 3.2 Known substitutions 9 EMBL outstation - the European Bioinformatics Institute. There are 3.3 Surface 9 no restrictions on its use as long as its content is in no way modified 3.4 Number of contacts 9 and this statement is not removed. 3.5 Annotation 9 3.6 Mutation suggestions 9 2.2 Multiple sequence alignment for 1aj8A 4 Appendix 9 For the chain 1aj8A, the alignment 1aj8A.msf (attached) with 309 4.1 File formats 9 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 9 database, and fragments shorter than 75% of the query as well as 4.3 Credits 9 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 9 to this report, under the name of 1aj8A.msf. Its statistics, from the 4.3.2 CE 10 alistat program are the following:

1 Lichtarge lab 2006 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. 1. Residues 6-190 in 1aj8A colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 2. Residues 191-376 in 1aj8A colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 3. Residues in 1aj8A, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 309 Total number of residues: 104073 Smallest: 174 Largest: 371 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Average length: 336.8 top 25% of all residues, this time colored according to clusters they Alignment length: 371 belong to. The clusters in Fig.4 are composed of the residues listed Average identity: 42% in Table 1. Most related pair: 99% Most unrelated pair: 11% Table 1. Most distant seq: 35% cluster size member color residues red 78 9,18,23,30,32,34,52,53,93,94 Furthermore, <1% of residues show as conserved in this ali- 95,177,179,181,182,184,185 gnment. 186,187,188,189,190,191,192 The alignment consists of 4% eukaryotic ( <1% arthropoda, 193,194,195,197,198,199,202 1% plantae), 89% prokaryotic, and 6% archaean sequences. (Des- 203,214,217,218,219,220,221 criptions of some sequences were not readily available.) The file 222,223,224,225,226,227,228 containing the sequence descriptions can be found in the attachment, 230,258,259,260,261,262,263 under the name 1aj8A.descr. 264,265,269,270,271,272,295 305,307,309,310,311,312,313 2.3 Residue ranking in 1aj8A 314,315,316,319,329,330,333 The 1aj8A sequence is shown in Figs. 1–2, with each residue colored 337,340,341,344,347 according to its estimated importance. The full listing of residues blue 8 130,133,137,151,160,161,162 in 1aj8A can be found in the file called 1aj8A.ranks sorted in the 164 attachment. yellow 3 206,208,209 green 2 356,357 2.4 Top ranking residues in 1aj8A and their position on the structure Table 1. Clusters of top ranking residues in 1aj8A. In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1aj8A colored by their

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 269 D D(93) 0.05 1/0 4.84 .(5)GS 312 D D(90) 0.05 8/0 3.38 site .(5) E(3) 337 R R(78) 0.09 10/0 2.80 .(21)K 263 R R(80) 0.10 2/1 4.64 .(5) A(7) P(6)K 333 F F(78) 0.12 13/0 3.79 .(19)LS TQ 311 V V(71) 0.15 6/0 3.80 .(5) L(12)P I(4) T(2) Fig. 4. Residues in 1aj8A, colored according to the cluster they belong to: A(3) red, followed by blue and yellow are the largest clusters (see Appendix for 222 I S(1) 0.21 2/2 4.22 the coloring scheme). Clockwise: front, back, top and bottom views. The A(41) corresponding Pymol script is attached. .(5) L(41)V K(6)IT 2.4.2 Overlap with known functional surfaces at 25% coverage. R(1) The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Table 2. The top 25% of residues in 1aj8A at the interface with citric . Table 2 lists the top 25% of residues at the acid.(Field names: res: residue number in the PDB entry; type: amino acid interface with 1aj8CIT1000 (citric acid). The following table (Table type; substs: substitutions seen in the alignment; with the percentage of each 3) suggests possible disruptive replacements for these residues (see type in the bracket; noc/bb: number of contacts with the ligand, with the num- Section 3.6). ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 2. res type subst’s cvg noc/ dist antn ˚ (%) bb (A) Table 3. 188 H H(99)Q 0.00 32/2 2.81 res type disruptive 191 N N(93) 0.01 28/6 3.14 mutations P(5)T 188 H (TE)(D)(SVMCAG)(QLPI) 271 R R(94) 0.02 19/0 2.75 191 N (Y)(H)(FW)(R) .(5) 271 R (TD)(SVCLAPIG)(YE)(FMW) 224 G G(94) 0.03 4/4 3.59 224 G (R)(KE)(H)(FWD) .(5)P 262 H (E)(T)(D)(M) 262 H H(93) 0.03 30/0 2.96 site 223 H (T)(EQ)(CG)(VA) .(5)N 310 N (Y)(FWH)(TR)(EVCAG) 223 H H(93) 0.04 31/9 2.98 site 192 A (YER)(K)(H)(D) .(5)DIE 269 D (R)(FWH)(K)(Y) 310 N N(94) 0.04 4/0 4.15 312 D (R)(FW)(H)(VCAG) .(5)S 337 R (T)(D)(Y)(VCAG) 192 A A(83) 0.05 1/1 4.98 263 R (T)(YD)(SE)(CG) C(10) 333 F (K)(E)(D)(QR) V(1) 311 V (R)(Y)(K)(E) N(4) 222 I (Y)(R)(H)(T) continued in next column continued in next column

3 Table 3. continued Table 4. continued res type disruptive res type subst’s cvg noc/ dist antn mutations (%) bb (A˚ ) .(5)SMF Table 3. List of disruptive mutations for the top 25% of residues in 1aj8A, R that are at the interface with citric acid. 310 N N(94) 0.04 14/0 3.04 .(5)S 312 D D(90) 0.05 1/0 4.02 site .(5) E(3) 261 G G(93) 0.06 26/26 3.37 .(5)VDS 260 A F(88) 0.10 30/28 2.84 .(5)YA I(1)M V(1)L 263 R R(80) 0.10 30/11 2.65 .(5) A(7) P(6)K 307 I L(73) 0.11 28/0 3.37 .(5) P(1) M(5) I(12)V 221 P P(90) 0.13 4/4 4.22 .(1) S(1) H(1)AVR NKD 258 M M(78) 0.14 19/10 3.62 Fig. 5. Residues in 1aj8A, at the interface with citric acid, colored by their .(5) relative importance. The ligand (citric acid) is colored green. Atoms further S(2)A A˚ than 30 away from the geometric center of the ligand, as well as on the line P(2) of sight to the ligand were removed. (See Appendix for the coloring scheme I(7) for the protein chain 1aj8A.) Y(1)FWN 222 I S(1) 0.21 34/20 2.96 Figure 5 shows residues in 1aj8A colored by their importance, at the A(41) interface with 1aj8CIT1000. .(5) binding site. Table 4 lists the top 25% of residues at L(41)V the interface with 1aj8COA3000 (coenzyme a). The following table K(6)IT (Table 5) suggests possible disruptive replacements for these residues R(1) (see Section 3.6). 305 K R(36) 0.21 23/0 3.04 K(54) Table 4. .(5)NQ res type subst’s cvg noc/ dist antn T(1)H (%) bb (A˚ ) 259 G G(94) 0.02 10/10 2.74 Table 4. The top 25% of residues in 1aj8A at the interface with coenzyme .(5) a.(Field names: res: residue number in the PDB entry; type: amino acid type; 224 G G(94) 0.03 1/1 4.87 substs: substitutions seen in the alignment; with the percentage of each type .(5)P in the bracket; noc/bb: number of contacts with the ligand, with the number of 262 H H(93) 0.03 17/16 3.70 site contacts realized through backbone atoms given in the bracket; dist: distance .(5)N of closest apporach to the ligand. ) 223 H H(93) 0.04 7/7 3.00 site .(5)DIE 226 A A(93) 0.04 8/3 3.26 continued in next column

4 Table 5. res type disruptive mutations 259 G (KER)(FQMWHD)(NLPI)(Y) 224 G (R)(KE)(H)(FWD) 262 H (E)(T)(D)(M) 223 H (T)(EQ)(CG)(VA) 226 A (E)(Y)(K)(R) 310 N (Y)(FWH)(TR)(EVCAG) 312 D (R)(FW)(H)(VCAG) 261 G (R)(K)(EH)(Q) 260 A (R)(K)(E)(Y) 263 R (T)(YD)(SE)(CG) 307 I (Y)(R)(H)(T) 221 P (Y)(R)(T)(H) 258 M (Y)(T)(R)(H) 222 I (Y)(R)(H)(T) 305 K (Y)(FTW)(VA)(CG)

Table 5. List of disruptive mutations for the top 25% of residues in 1aj8A, that are at the interface with coenzyme a.

Fig. 6. Residues in 1aj8A, at the interface with coenzyme a, colored by their relative importance. The ligand (coenzyme a) 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 1aj8A.)

Figure 6 shows residues in 1aj8A colored by their importance, at the interface with 1aj8COA3000. Citric acid binding site. Table 6 lists the top 25% of residues at the interface with 1aj8CIT2000 (citric acid). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

5 Table 6. Table 8. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist antn (%) bb (A˚ ) (%) bb (A˚ ) 356 R R(77) 0.14 16/0 2.63 188 H H(99)Q 0.00 7/4 3.59 .(21)GS 191 N N(93) 0.01 23/17 3.61 L P(5)T 220 G G(98) 0.01 27/27 3.17 Table 6. The top 25% of residues in 1aj8A at the interface with citric .(1)I acid.(Field names: res: residue number in the PDB entry; type: amino acid 262 H H(93) 0.03 2/0 4.23 site type; substs: substitutions seen in the alignment; with the percentage of each .(5)N type in the bracket; noc/bb: number of contacts with the ligand, with the num- 223 H H(93) 0.04 16/5 3.81 site ber of contacts realized through backbone atoms given in the bracket; dist: .(5)DIE distance of closest apporach to the ligand. ) 192 A A(83) 0.05 23/14 3.45 C(10) V(1) Table 7. N(4) res type disruptive 202 S S(88) 0.06 5/4 3.31 mutations A(5) 356 R (D)(TYE)(LPI)(FVCAWG) G(5)T 198 M R(89) 0.07 4/0 4.31 Table 7. List of disruptive mutations for the top 25% of residues in 1aj8A, I(1) that are at the interface with citric acid. M(1) H(2) L(3)VQ 206 D N(50) 0.08 18/2 2.72 G(2) D(42) S(2) H(1)E 189 E E(82) 0.09 66/4 2.45 G(10) T(1) S(4)D 195 L S(46) 0.10 22/1 3.49 A(10) F(34) H(1)L T(6)N 263 R R(80) 0.10 68/1 2.76 .(5) A(7) P(6)K 203 T S(47) 0.11 66/33 2.88 T(42) A(9) 34 G G(87) 0.12 48/48 2.80 Fig. 7. Residues in 1aj8A, at the interface with citric acid, colored by their .(10)DA relative importance. The ligand (citric acid) is colored green. Atoms further QE than 30A˚ away from the geometric center of the ligand, as well as on the line 221 P P(90) 0.13 28/7 3.25 of sight to the ligand were removed. (See Appendix for the coloring scheme .(1) for the protein chain 1aj8A.) S(1) H(1)AVR continued in next column Figure 7 shows residues in 1aj8A colored by their importance, at the interface with 1aj8CIT2000. Interface with 1aj8B.Table 8 lists the top 25% of residues at the interface with 1aj8B. The following table (Table 9) suggests possible disruptive replacements for these residues (see Section 3.6).

6 Table 8. continued Table 8. continued res type subst’s cvg noc/ dist antn res type subst’s cvg noc/ dist antn (%) bb (A˚ ) (%) bb (A˚ ) NKD R(1) 9 G G(71) 0.14 52/52 2.92 93 H H(82)NS 0.22 20/2 3.15 .(28)A .(6)T 264 V L(1) 0.14 20/0 3.51 D(5)PQR V(84) EGLA .(5) 199 T L(38) 0.22 21/2 3.33 I(6)F M(5) E(1)A T(3) 356 R R(77) 0.14 133/35 2.34 I(8) .(21)GS V(35) L A(1)SC 357 P P(77) 0.15 76/33 3.26 N(1) .(21)HR H(2)FQ Q 18 T S(57) 0.23 66/40 2.80 190 I Q(58) 0.16 79/34 3.13 .(14) L(10) T(26)KY G(2) 23 I V(20) 0.23 12/6 3.16 F(15) I(62) M(8) .(11) I(2) L(5)PR V(2) 217 A C(21) 0.23 31/23 2.92 209 S A(58) 0.16 31/9 2.81 T(18) S(30) S(19) V(1) A(38) L(1)N G(1). G(2) 344 H Q(24) 0.24 5/0 3.41 T(4)H .(21) 347 E E(75) 0.16 1/0 4.82 H(51) .(22) S(1) D(1)A N(1) 59 P P(88) 0.17 27/22 2.70 354 I I(59) 0.25 35/18 2.93 .(9)NSE .(21) KDA L(18)VN 219 K W(52) 0.18 42/4 2.74 K .(1) K(21) Table 8. The top 25% of residues in 1aj8A at the interface with 1aj8B. S(5) (Field names: res: residue number in the PDB entry; type: amino acid type; A(3) substs: substitutions seen in the alignment; with the percentage of each type R(9) in the bracket; noc/bb: number of contacts with the ligand, with the number of Y(3)TMG contacts realized through backbone atoms given in the bracket; dist: distance F of closest apporach to the ligand. ) 208 Y Y(39) 0.19 12/8 3.80 F(49) Table 9. H(4) I(1)V res type disruptive A(1)T mutations L(1)SR 188 H (TE)(D)(SVMCAG)(QLPI) 222 I S(1) 0.21 40/6 3.25 191 N (Y)(H)(FW)(R) A(41) 220 G (R)(KE)(H)(FWD) .(5) 262 H (E)(T)(D)(M) L(41)V 223 H (T)(EQ)(CG)(VA) K(6)IT 192 A (YER)(K)(H)(D) 202 S (KR)(QH)(FMW)(E) continued in next column continued in next column

7 Table 9. continued res type disruptive mutations 198 M (Y)(T)(H)(CG) 206 D (R)(FW)(H)(K) 189 E (FWH)(R)(Y)(VA) 195 L (R)(Y)(KE)(H) 263 R (T)(YD)(SE)(CG) 203 T (KR)(QH)(FMW)(E) 34 G (R)(H)(FW)(K) 221 P (Y)(R)(T)(H) 9 G (KER)(HD)(Q)(FMW) 264 V (R)(Y)(K)(E) 356 R (D)(TYE)(LPI)(FVCAWG) 357 P (Y)(T)(SR)(CG) 190 I (Y)(R)(H)(T) 209 S (R)(K)(QH)(E) 347 E (H)(FW)(R)(Y) 59 P (Y)(R)(H)(T) 219 K (Y)(T)(FW)(D) 208 Y (K)(Q)(E)(MR) 222 I (Y)(R)(H)(T) 93 H (E)(T)(MD)(Q) Fig. 8. Residues in 1aj8A, at the interface with 1aj8B, colored by their rela- 199 T (R)(K)(H)(EQ) tive importance. 1aj8B is shown in backbone representation (See Appendix 18 T (KR)(FMW)(Q)(H) for the coloring scheme for the protein chain 1aj8A.) 23 I (Y)(R)(T)(H) 217 A (KR)(E)(Q)(YH) 344 H (E)(T)(MD)(QCG) Table 11. 354 I (Y)(R)(TH)(SECG) res type disruptive mutations Table 9. List of disruptive mutations for the top 25% of residues in 1aj8A, 354 I (Y)(R)(TH)(SECG) that are at the interface with 1aj8B. Table 11. List of disruptive mutations for the top 25% of residues in Figure 8 shows residues in 1aj8A colored by their importance, at the 1aj8A, that are at the interface with coenzyme a. interface with 1aj8B. Coenzyme a binding site. Table 10 lists the top 25% of residues Figure 9 shows residues in 1aj8A colored by their importance, at the at the interface with 1aj8COA4000 (coenzyme a). The following interface with 1aj8COA4000. table (Table 11) suggests possible disruptive replacements for these residues (see Section 3.6). 3 NOTES ON USING TRACE RESULTS Table 10. res type subst’s cvg noc/ dist 3.1 Coverage (%) bb (A˚ ) Trace results are commonly expressed in terms of coverage: the resi- 354 I I(59) 0.25 13/2 3.92 due is important if its “coverage” is small - that is if it belongs to .(21) some small top percentage of residues [100% is all of the residues L(18)VN in a chain], according to trace. The ET results are presented in the K form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed Table 10. The top 25% of residues in 1aj8A at the interface with coen- evolutionary pressure. (I.e., the smaller the coverage, the stronger the zyme a.(Field names: res: residue number in the PDB entry; type: amino acid pressure on the residue.) Starting from the top of that list, mutating a type; substs: substitutions seen in the alignment; with the percentage of each couple of residues should affect the protein somehow, with the exact type in the bracket; noc/bb: number of contacts with the ligand, with the num- effects to be determined experimentally. ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types 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 example if the substitutions are “RVK” and the original protein has

8 3.5 Annotation If the residue annotation is available (either from the pdb file or 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 bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following properties: small [AV GSTC], medium [LPNQDEMIK], large [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- tively [KHR], or negatively [DE] charged, aromatic [WFYH], long aliphatic chain [EKRQM], OH-group possession [SDETY ], and NH2 group possession [NQRK]. The suggestions are listed according to how different they appear to be from the original amino acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less Fig. 9. Residues in 1aj8A, at the interface with coenzyme a, colored by their disruptive) These suggestions are tentative - they might prove disrup- relative importance. The ligand (coenzyme a) is colored green. Atoms further tive to the fold rather than to the interaction. Many researcher will than 30A˚ away from the geometric center of the ligand, as well as on the line choose, however, the straightforward alanine mutations, especially in of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1aj8A.) the beginning stages of their investigation.

4 APPENDIX 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 to 100%. amino acid type • 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, mutation presumably won’t have strong impact). Two heavy atoms DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, are considered to be “in contact” if their centers are closer than 5A˚ . tan, DarkOrange, DeepPink, maroon, BlanchedAlmond.

9 http://swift.cmbi.kun.nl/swift/hssp/

4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). COVERAGE 4.3.6 Muscle When making alignments “from scratch”, report

V maker uses Muscle alignment program: Edgar, Robert C. (2004), 100% 50% 30% 5% ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. http://www.drive5.com/muscle/

4.3.7 Pymol The figures in this report were produced using

V Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- 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 Fig. 10. Coloring scheme used to color residues by their relative importance. the scripts and launch the viewer.) 4.4 Note about ET Viewer The colors used to distinguish the residues by the estimated Dan Morgan from the Lichtarge lab has developed a visualization evolutionary pressure they experience can be seen in Fig. 10. 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 http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- 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- 4.5 Citing this work ned as (idents / MIN(len1, len2)) where idents is the number of 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 of service for comparative analysis of .” Bioinformatics 4.3.2 CE To map ligand binding sites from different 22:1656-7. source structures, report maker uses the CE program: http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension report maker was written in 2006 by Ivana Mihalek. The 1D ran- (CE) of the optimal path . Protein Engineering 11(9) 739-747. king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 Houston. ble if the DSSP program finds it exposed to water by at least 10A˚ , which is roughly the area needed for one water molecule to come in 4.7 Attachments the contact with the residue. DSSP is copyrighted by W. Kabsch, C. The following files should accompany this report: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, • 1aj8A.complex.pdb - coordinates of 1aj8A with all of its inter- acting partners http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1aj8A.etvx - ET viewer input file for 1aj8A 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • 1aj8A.cluster report.summary - Cluster report summary for gnment as a starting point for the analysis (sequences shorter than 1aj8A 75% of the query are taken out, however); R. Schneider, A. de • Daruvar, and C. Sander. ”The HSSP database of protein structure- 1aj8A.ranks - Ranks file in sequence order for 1aj8A sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 1aj8A.clusters - Cluster descriptions for 1aj8A

10 • 1aj8A.msf - the multiple sequence alignment used for the chain • 1aj8A.cbcvg - used by other 1aj8A – related pymol scripts 1aj8A • 1aj8A.1aj8COA3000.if.pml - Pymol script for Figure 6 • 1aj8A.descr - description of sequences used in 1aj8A msf • 1aj8A.1aj8CIT2000.if.pml - Pymol script for Figure 7 • 1aj8A.ranks sorted - full listing of residues and their ranking for • 1aj8A.1aj8B.if.pml - Pymol script for Figure 8 1aj8A • 1aj8A.1aj8COA4000.if.pml - Pymol script for Figure 9 • 1aj8A.1aj8CIT1000.if.pml - Pymol script for Figure 5

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