Pages 1–9 2phd Evolutionary trace report by report maker January 27, 2010

4.3.1 Alistat 8 4.3.2 CE 8 4.3.3 DSSP 8 4.3.4 HSSP 9 4.3.5 LaTex 9 4.3.6 Muscle 9 4.3.7 Pymol 9 4.4 Note about ET Viewer 9 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 2phd): Title: Crystal structure determination of a salicylate 1,2- dioxygenase from pseudaminobacter salicylatoxidans CONTENTS Compound: Mol id: 1; molecule: gentisate 1,2-dioxygenase; chain: a, b, c, d; ec: 1.13.11.4; engineered: yes 1 Introduction 1 Organism, scientific name: Pseudaminobacter Salicylatoxidans; 2phd contains a single unique chain 2phdD (352 residues long) and 2 Chain 2phdD 1 its homologues 2phdA, 2phdC, and 2phdB. 2.1 Q67FT0 overview 1 2.2 Multiple sequence alignment for 2phdD 1 2.3 Residue ranking in 2phdD 1 2.4 Top ranking residues in 2phdD and their position on the structure 1 2.4.1 Clustering of residues at 25% coverage. 1 2 CHAIN 2PHDD 2.4.2 Overlap with known functional surfaces at 2.1 Q67FT0 overview 25% coverage. 2 2.4.3 Possible novel functional surfaces at 25% From SwissProt, id Q67FT0, 94% identical to 2phdD: coverage. 6 Description: Gentisate 1,2-dioxygenase. Organism, scientific name: Pseudaminobacter salicylatoxidans. 3 Notes on using trace results 7 Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Rhizo- 3.1 Coverage 7 biales; Phyllobacteriaceae; Pseudaminobacter. 3.2 Known substitutions 7 3.3 Surface 7 3.4 Number of contacts 8 3.5 Annotation 8 2.2 Multiple sequence alignment for 2phdD 3.6 Mutation suggestions 8 For the chain 2phdD, the alignment 2phdD.msf (attached) with 102 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 8 database, and fragments shorter than 75% of the query as well as 4.1 File formats 8 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 8 to this report, under the name of 2phdD.msf. Its statistics, from the 4.3 Credits 8 alistat program are the following:

1 Lichtarge lab 2006 by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 15-190 in 2phdD colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.)

Fig. 2. Residues 191-366 in 2phdD colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.)

Fig. 3. Residues in 2phdD, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 102 Total number of residues: 33659 Smallest: 114 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 352 top 25% of all residues, this time colored according to clusters they Average length: 330.0 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 352 in Table 1. Average identity: 38% Most related pair: 99% Table 1. Most unrelated pair: 11% cluster size member Most distant seq: 32% color residues red 76 59,72,81,83,84,85,88,90,91 100,103,104,106,108,109,112 Furthermore, <1% of residues show as conserved in this ali- 113,114,116,117,119,120,121 gnment. 123,124,125,126,127,128,129 The alignment consists of <1% eukaryotic ( <1% fungi), 23% 131,132,134,135,137,139,141 prokaryotic, and <1% archaean sequences. (Descriptions of some 143,146,149,150,151,152,153 sequences were not readily available.) The file containing the 154,155,157,159,160,161,162 sequence descriptions can be found in the attachment, under the name 164,169,170,172,173,174,175 2phdD.descr. 176,177,178,179,188,228,229 235,268,269,272,305,308,325 2.3 Residue ranking in 2phdD 345,346,350,352 The 2phdD sequence is shown in Figs. 1–2, with each residue colored blue 4 50,298,330,332 according to its estimated importance. The full listing of residues yellow 3 37,38,39 in 2phdD can be found in the file called 2phdD.ranks sorted in the attachment. Table 1. Clusters of top ranking residues in 2phdD. 2.4 Top ranking residues in 2phdD and their position on the structure 2.4.2 Overlap with known functional surfaces at 25% coverage. In the following we consider residues ranking among top 25% of The name of the ligand is composed of the source PDB identifier residues in the protein . Figure 3 shows residues in 2phdD colored and the heteroatom name used in that file.

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) .(3) 104 W Y(72) 0.13 9/0 4.37 W(13) F(2) N(3)L .(1) V(1)SA 332 W Y(5) 0.13 18/0 3.81 W(80) F(4) N(1) H(2) .(1) D(1) 123 Q P(16) 0.14 17/3 3.81 Q(52) A(20) M(5) S(1)VT Fig. 4. Residues in 2phdD, colored according to the cluster they belong to: 188 F Y(15) 0.15 107/19 3.31 red, followed by blue and yellow are the largest clusters (see Appendix for F(79)SV the coloring scheme). Clockwise: front, back, top and bottom views. The .RD corresponding Pymol script is attached. 357 L L(89)MA 0.15 27/6 3.52 .(1) F(3) Interface with 2phdB.Table 2 lists the top 25% of residues at the V(1)I interface with 2phdB. The following table (Table 3) suggests possible 88 L L(88) 0.16 4/3 3.87 disruptive replacements for these residues (see Section 3.6). F(6) Table 2. M(1).VP res type subst’s cvg noc/ dist antn 178 I L(33) 0.16 14/0 3.13 (%) bb (A˚ ) I(56) 121 H H(99)N 0.03 9/0 4.04 site V(5)H 176 L L(99). 0.03 2/0 4.39 A(1). 177 D D(99). 0.03 6/0 3.67 85 A V(74) 0.17 31/28 2.78 59 W W(98) 0.06 30/0 3.21 A(14) .(1) N(5).QH 352 P P(93) 0.07 2/2 4.70 KT V(1) 157 W G(26) 0.18 20/0 3.56 A(1)D W(63) .(1) N(1) 84 R R(98).F 0.09 10/5 3.96 M(5)FS 91 P P(96).G 0.09 7/2 3.50 37 P P(77) 0.19 12/10 3.63 TD A(13) 83 R R(96) 0.10 80/22 2.88 G(4) G(1).T .(3) 39 W W(95) 0.11 92/10 3.03 81 G A(66) 0.21 1/1 4.95 .(3)F G(22) 50 P P(94)E 0.11 39/12 3.04 T(4) .(2)VG .(2)PDF 38 L L(89) 0.12 40/15 2.93 298 V S(14) 0.22 11/2 3.70 Q(4) T(60) G(1) V(17) continued in next column continued in next column

3 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) N(3)A .(1) 124 N N(26) 0.25 3/0 4.09 S(51) F(8) A(4) C(1) G(2)HET

Table 2. The top 25% of residues in 2phdD at the interface with 2phdB. (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. )

Table 3. res type disruptive mutations Fig. 5. Residues in 2phdD, at the interface with 2phdB, colored by their rela- 121 H (E)(T)(MD)(SVQCAG) tive importance. 2phdB is shown in backbone representation (See Appendix 176 L (YR)(TH)(SCG)(KE) for the coloring scheme for the protein chain 2phdD.) 177 D (R)(FWH)(VCAG)(KY) 59 W (KE)(TQD)(SNCG)(R) 352 P (R)(Y)(H)(K) Table 4. 84 R (TD)(SECG)(VLAPI)(Y) res type subst’s cvg noc/ dist antn 91 P (R)(Y)(H)(K) (%) bb (A˚ ) 83 R (D)(ELPI)(FTYVMAW)(S) 119 H H(100) 0.01 5/0 2.02 site 39 W (E)(K)(TD)(Q) 160 H H(100) 0.01 5/0 2.16 site 50 P (R)(Y)(H)(TK) 121 H H(99)N 0.03 5/0 2.13 site 38 L (Y)(R)(H)(T) 127 R R(99)Q 0.03 1/0 4.73 104 W (K)(E)(Q)(D) 332 W (K)(E)(TQ)(D) Table 4. The top 25% of residues in 2phdD at the interface with fe (iii) 123 Q (Y)(H)(FW)(T) ion.(Field names: res: residue number in the PDB entry; type: amino acid 188 F (K)(E)(Q)(D) type; substs: substitutions seen in the alignment; with the percentage of each 357 L (YR)(TH)(K)(E) type in the bracket; noc/bb: number of contacts with the ligand, with the num- 88 L (YR)(T)(H)(KE) ber of contacts realized through backbone atoms given in the bracket; dist: 178 I (R)(Y)(T)(EH) distance of closest apporach to the ligand. ) 85 A (Y)(E)(R)(K) 157 W (KE)(D)(Q)(TR) 37 P (R)(Y)(H)(KE) 81 G (R)(K)(E)(Q) Table 5. 298 V (R)(K)(YE)(H) res type disruptive 124 N (Y)(R)(H)(FW) mutations 119 H (E)(TQMD)(SNKVCLAPIG)(YR) 160 H (E)(TQMD)(SNKVCLAPIG)(YR) Table 3. List of disruptive mutations for the top 25% of residues in 2phdD, that are at the interface with 2phdB. 121 H (E)(T)(MD)(SVQCAG) 127 R (T)(YD)(SVCAG)(FELWPI)

Figure 5 shows residues in 2phdD colored by their importance, at the Table 5. List of disruptive mutations for the top 25% of residues in interface with 2phdB. 2phdD, that are at the interface with fe (iii) ion. Fe (iii) ion binding site. Table 4 lists the top 25% of residues at the interface with 2phdDFE370 (fe (iii) ion). The following table (Table 5) suggests possible disruptive replacements for these residues (see Figure 6 shows residues in 2phdD colored by their importance, at the Section 3.6). interface with 2phdDFE370. Acetate ion binding site. Table 6 lists the top 25% of residues at the interface with 2phdDACT1 (acetate ion). The following table

4 Fig. 6. Residues in 2phdD, at the interface with fe (iii) ion, colored by their Fig. 7. Residues in 2phdD, at the interface with acetate ion, colored by their relative importance. The ligand (fe (iii) ion) is colored green. Atoms further relative importance. The ligand (acetate ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line 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 of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 2phdD.) for the protein chain 2phdD.)

(Table 7) suggests possible disruptive replacements for these residues Interface with 2phdA.Table 8 lists the top 25% of residues at the (see Section 3.6). interface with 2phdA. The following table (Table 9) suggests 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˚ ) 38 L L(89) 0.12 4/0 3.69 272 G G(94) 0.12 26/26 2.80 Q(4) K(1)PA G(1) .(1) .(3) 218 P P(81)A 0.15 22/17 3.48 R(3) Table 6. The top 25% of residues in 2phdD at the interface with acetate .(8) ion.(Field names: res: residue number in the PDB entry; type: amino acid V(2)GL type; substs: substitutions seen in the alignment; with the percentage of each 269 P P(88) 0.20 2/2 3.96 type in the bracket; noc/bb: number of contacts with the ligand, with the num- G(1) ber of contacts realized through backbone atoms given in the bracket; dist: .(3) distance of closest apporach to the ligand. ) S(1) L(1)RA

Table 7. Table 8. The top 25% of residues in 2phdD at the interface with 2phdA. res type disruptive (Field names: res: residue number in the PDB entry; type: amino acid type; mutations substs: substitutions seen in the alignment; with the percentage of each type 38 L (Y)(R)(H)(T) 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 7. List of disruptive mutations for the top 25% of residues in 2phdD, that are at the interface with acetate ion.

Figure 7 shows residues in 2phdD colored by their importance, at the interface with 2phdDACT1.

5 Table 9. Table 10. continued res type disruptive res type subst’s cvg noc/ dist antn mutations (%) bb (A˚ ) 272 G (E)(R)(H)(K) F(2) 218 P (Y)(R)(H)(E) N(3)L 269 P (Y)(R)(H)(TE) .(1) V(1)SA Table 9. List of disruptive mutations for the top 25% of residues in 178 I L(33) 0.16 1/0 4.98 2phdD, that are at the interface with 2phdA. I(56) V(5)H A(1).

Table 10. The top 25% of residues in 2phdD at the interface with acetate ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each type in the bracket; noc/bb: number of contacts with the ligand, with the num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 11. res type disruptive mutations 119 H (E)(TQMD)(SNKVCLAPIG)(YR) 160 H (E)(TQMD)(SNKVCLAPIG)(YR) 121 H (E)(T)(MD)(SVQCAG) 174 D (R)(FWH)(VCAG)(KY) 176 L (YR)(TH)(SCG)(KE) 104 W (K)(E)(Q)(D) 178 I (R)(Y)(T)(EH)

Table 11. List of disruptive mutations for the top 25% of residues in 2phdD, that are at the interface with acetate ion.

Fig. 8. Residues in 2phdD, at the interface with 2phdA, colored by their rela- Figure 9 shows residues in 2phdD colored by their importance, at the tive importance. 2phdA is shown in backbone representation (See Appendix interface with 2phdAACT371. for the coloring scheme for the protein chain 2phdD.) 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2phdD surface, away from (or Figure 8 shows residues in 2phdD colored by their importance, at the susbtantially larger than) other functional sites and interfaces reco- interface with 2phdA. gnizable in PDB entry 2phd. It is shown in Fig. 10. The right panel Acetate ion binding site. By analogy with 2phdA – shows (in blue) the rest of the larger cluster this surface belongs to. 2phdAACT371 interface. Table 10 lists the top 25% of residues The residues belonging to this surface ”patch” are listed in Table 12, at the interface with 2phdAACT371 (acetate ion). The following while Table 13 suggests possible disruptive replacements for these table (Table 11) suggests possible disruptive replacements for these residues (see Section 3.6). residues (see Section 3.6). Table 12. Table 10. res type substitutions(%) cvg res type subst’s cvg noc/ dist antn 352 P P(93)V(1)A(1)D 0.07 (%) bb (A˚ ) .(1) 119 H H(100) 0.01 3/0 4.45 site 350 D E(9)D(87).(1)N 0.08 160 H H(100) 0.01 1/0 4.91 site 50 P P(94)E.(2)VG 0.11 121 H H(99)N 0.03 5/0 4.37 site 332 W Y(5)W(80)F(4) 0.13 174 D D(99). 0.03 10/0 4.06 N(1)H(2).(1) 176 L L(99). 0.03 9/0 3.71 D(1) 104 W Y(72) 0.13 3/0 4.15 357 L L(89)MA.(1)F(3) 0.15 W(13) V(1)I continued in next column continued in next column

6 Table 13. continued res type disruptive mutations 50 P (R)(Y)(H)(TK) 332 W (K)(E)(TQ)(D) 357 L (YR)(TH)(K)(E) 298 V (R)(K)(YE)(H)

Table 13. Disruptive mutations for the surface patch in 2phdD.

Another group of surface residues is shown in Fig.11. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

Fig. 9. Residues in 2phdD, at the interface with acetate ion, colored by their relative importance. The ligand (acetate 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 2phdD.) Fig. 11. Another possible active surface on the chain 2phdD. The larger cluster it belongs to is shown in blue.

The residues belonging to this surface ”patch” are listed in Table 14, while Table 15 suggests possible disruptive replacements for these residues (see Section 3.6).

Table 14. res type substitutions(%) cvg 325 D D(98).(1) 0.04 59 W W(98).(1) 0.06 91 P P(96).GTD 0.09 Fig. 10. A possible active surface on the chain 2phdD. The larger cluster it 90 N N(94)D(2)S.V 0.10 belongs to is shown in blue. 88 L L(88)F(6)M(1).V 0.16 P

Table 12. continued Table 14. Residues forming surface ”patch” in 2phdD. res type substitutions(%) cvg 298 V S(14)T(60)V(17) 0.22 N(3)A.(1) Table 15. Table 12. Residues forming surface ”patch” in 2phdD. res type disruptive mutations 325 D (R)(FWH)(VCAG)(KY) Table 13. 59 W (KE)(TQD)(SNCG)(R) res type disruptive 91 P (R)(Y)(H)(K) mutations 90 N (Y)(H)(FW)(R) 352 P (R)(Y)(H)(K) 88 L (YR)(T)(H)(KE) 350 D (R)(FWH)(YVCAG)(TK) continued in next column Table 15. Disruptive mutations for the surface patch in 2phdD.

7 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. 12. 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

8 ”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. 12. 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, • 2phdD.complex.pdb - coordinates of 2phdD with all of its http://www.cmbi.kun.nl/gv/dssp/descrip.html. interacting partners 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • 2phdD.etvx - ET viewer input file for 2phdD gnment as a starting point for the analysis (sequences shorter than • 2phdD.cluster report.summary - Cluster report summary for 75% of the query are taken out, however); R. Schneider, A. de 2phdD Daruvar, and C. Sander. ”The HSSP database of protein structure- • 2phdD.ranks - Ranks file in sequence order for 2phdD sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 2phdD.clusters - Cluster descriptions for 2phdD http://swift.cmbi.kun.nl/swift/hssp/ • 2phdD.msf - the multiple sequence alignment used for the chain 2phdD 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 2phdD.descr - description of sequences used in 2phdD msf Wesley,” Reading, Mass. (1986). • 2phdD.ranks sorted - full listing of residues and their ranking 4.3.6 Muscle When making alignments “from scratch”, report for 2phdD maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2phdD.2phdB.if.pml - Pymol script for Figure 5

9 • 2phdD.cbcvg - used by other 2phdD – related pymol scripts • 2phdD.2phdA.if.pml - Pymol script for Figure 8 • 2phdD.2phdDFE370.if.pml - Pymol script for Figure 6 • 2phdD.2phdAACT371.if.pml - Pymol script for Figure 9 • 2phdD.2phdDACT1.if.pml - Pymol script for Figure 7

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