Pages 1–10 1y75 Evolutionary trace report by report maker January 28, 2010

4 Notes on using trace results 7 4.1 Coverage 7 4.2 Known substitutions 7 4.3 Surface 8 4.4 Number of contacts 8 4.5 Annotation 8 4.6 Mutation suggestions 8

5 Appendix 8 5.1 File formats 8 5.2 Color schemes used 8 5.3 Credits 8 5.3.1 Alistat 8 5.3.2 CE 9 5.3.3 DSSP 9 5.3.4 HSSP 9 5.3.5 LaTex 9 5.3.6 Muscle 9 5.3.7 Pymol 9 5.4 Note about ET Viewer 9 5.5 Citing this work 9 CONTENTS 5.6 About report maker 9 5.7 Attachments 9 1 Introduction 1

2 Chain 1y75A 1 1 INTRODUCTION 2.1 Q5G291 overview 1 From the original Protein Data Bank entry (PDB id 1y75): 2.2 Multiple sequence alignment for 1y75A 1 Title: A new form of catalytically inactive phospholipase a2 with 2.3 Residue ranking in 1y75A 1 an unusual disulphide bridge cys 32- cys 49 reveals recognition for 2.4 Top ranking residues in 1y75A and their position on n-acetylglucosmine the structure 1 Compound: Mol id: 1; molecule: phospholipase a2 isoform 5; chain: 2.4.1 Clustering of residues at 25% coverage. 2 a; ec: 3.1.1.4; mol id: 2; molecule: phospholipase a2 isoform 6; 2.4.2 Overlap with known functional surfaces at chain: b; ec: 3.1.1.4 25% coverage. 3 Organism, scientific name: Naja Sagittifera; 2.4.3 Possible novel functional surfaces at 25% 1y75 contains unique chains 1y75A (118 residues) and 1y75B (118 coverage. 4 residues) 3 Chain 1y75B 5 3.1 Q5G290 overview 5 2 CHAIN 1Y75A 3.2 Multiple sequence alignment for 1y75B 5 3.3 Residue ranking in 1y75B 5 2.1 Q5G291 overview 3.4 Top ranking residues in 1y75B and their position on From SwissProt, id Q5G291, 100% identical to 1y75A: the structure 5 Description: Phospholipase A2 isoform 5 (Fragment). 3.4.1 Clustering of residues at 25% coverage. 5 Organism, scientific name: Naja sagittifera (Andaman cobra). 3.4.2 Overlap with known functional surfaces at Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 25% coverage. 6 Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; 3.4.3 Possible novel functional surfaces at 25% Colubroidea; Elapidae; Elapinae; Naja. coverage. 6 Similarity: Belongs to the phospholipase A2 family.

1 Lichtarge lab 2006 Fig. 1. Residues 1-120 in 1y75A colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.)

2.2 Multiple sequence alignment for 1y75A For the chain 1y75A, the alignment 1y75A.msf (attached) with 518 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1y75A.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 518 Fig. 2. Residues in 1y75A, colored by their relative importance. Clockwise: Total number of residues: 56626 front, back, top and bottom views. Smallest: 41 Largest: 118 Average length: 109.3 Alignment length: 118 Average identity: 44% Most related pair: 99% Most unrelated pair: 0% Most distant seq: 30%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 62% eukaryotic ( 60% vertebrata, <1% arthropoda, <1% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1y75A.descr. 2.3 Residue ranking in 1y75A The 1y75A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1y75A can be found in the file called 1y75A.ranks sorted in the attachment.

2.4 Top ranking residues in 1y75A and their position on Fig. 3. Residues in 1y75A, colored according to the cluster they belong to: the structure red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The In the following we consider residues ranking among top 25% of corresponding Pymol script is attached. residues in the protein . Figure 2 shows residues in 1y75A colored by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A in Table 1. Pymol script for producing this figure can be found in the attachment.

2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed

2 Table 1. cluster size member color residues red 27 5,9,22,25,26,27,28,29,30,33 35,37,39,41,42,44,45,48,49 51,52,68,79,91,93,94,100 blue 2 61,86

Table 1. Clusters of top ranking residues in 1y75A.

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 1y75B.Table 2 lists the top 25% of residues at the interface with 1y75B. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 4.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 30 G G(97)S. 0.06 2/2 4.51 EDNR Fig. 4. Residues in 1y75A, at the interface with 1y75B, colored by their rela- tive importance. 1y75B is shown in backbone representation (See Appendix Table 2. The top 25% of residues in 1y75A at the interface with 1y75B. for the coloring scheme for the protein chain 1y75A.) (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 Table 4. continued contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 22 F Y(87) 0.14 12/10 2.80 Table 3. F(10) res type disruptive .(1)LN mutations 52 Y Y(94)CV 0.17 3/0 4.54 30 G (R)(FW)(KH)(E) .FLSWHA 28 Y H(4) 0.21 5/5 3.50 Table 3. List of disruptive mutations for the top 25% of residues in N(7) 1y75A, that are at the interface with 1y75B. Y(81) F(2) W(3).ES Figure 4 shows residues in 1y75A colored by their importance, at the 5 F F(80) 0.22 33/2 3.42 interface with 1y75B. L(14) NAG binding site. Table 4 lists the top 25% of residues at the inter- .(4)MYE face with 1y75NAG301 (nag). The following table (Table 5) suggests 9 I I(88) 0.24 2/0 3.71 possible disruptive replacements for these residues (see Section 4.6). V(4) Table 4. .(3) res type subst’s cvg noc/ dist antn M(1) (%) bb (A˚ ) L(1)FNW 29 C C(99).A 0.01 15/11 3.65 S-S SA W 49 C D(85) 0.25 3/1 3.96 S-S 30 G G(97)S. 0.06 14/14 3.36 K(5)R EDNR .(1)CH 45 C C(98).R 0.08 12/5 3.72 S-S S(1)G YL N(1)EAQ 48 H H(97).R 0.10 14/2 3.09 M YSQN continued in next column Table 4. The top 25% of residues in 1y75A at the interface with NAG.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each

3 type in the bracket; noc/bb: number of contacts with the ligand, with the num- shows (in blue) the rest of the larger cluster this surface belongs to. ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 5. res type disruptive mutations 29 C (KE)(R)(QD)(MH) 30 G (R)(FW)(KH)(E) 45 C (E)(K)(R)(D) 48 H (E)(T)(MD)(VQA) 22 F (KE)(T)(Q)(D) 52 Y (K)(Q)(E)(M) 28 Y (K)(Q)(M)(ER) Fig. 6. A possible active surface on the chain 1y75A. The larger cluster it 5 F (K)(TE)(QR)(CDG) belongs to is shown in blue. 9 I (R)(Y)(T)(H) 49 C (R)(E)(FW)(KH) The residues belonging to this surface ”patch” are listed in Table Table 5. List of disruptive mutations for the top 25% of residues in 6, while Table 7 suggests possible disruptive replacements for these 1y75A, that are at the interface with NAG. residues (see Section 4.6). Table 6. res type substitutions(%) cvg antn 29 C C(99).AW 0.01 S-S 39 D D(98).QREPG 0.02 27 C C(96)R.N(2)KDE 0.03 S-S 51 C C(99).F 0.04 S-S 26 G G(98).FASL 0.05 30 G G(97)S.EDNR 0.06 45 C C(98).RYL 0.08 S-S 33 G G(98).PRNSKA 0.09 44 C C(98).ATYWG 0.09 S-S 48 H H(97).RYSQN 0.10 68 Y Y(94)F.(1)CSLRI 0.12 AHG 22 F Y(87)F(10).(1)L 0.14 N 35 G G(93)KR(2).ENQL 0.14 HIPASD 93 C C(96).(2)AYL 0.15 S-S 37 P P(95).A(2)TRKIG 0.16 Y 52 Y Y(94)CV.FLSWHA 0.17 100 C C(95).(3)RYV 0.18 S-S 28 Y H(4)N(7)Y(81) 0.21 F(2)W(3).ES Fig. 5. Residues in 1y75A, at the interface with NAG, colored by their relative 5 F F(80)L(14).(4)M 0.22 A˚ importance. The ligand (NAG) is colored green. Atoms further than 30 away YE from the geometric center of the ligand, as well as on the line of sight to the 9 I I(88)V(4).(3) 0.24 ligand were removed. (See Appendix for the coloring scheme for the protein chain 1y75A.) M(1)L(1)FNWSA 49 C D(85)K(5)R.(1)C 0.25 S-S HS(1)GN(1)EAQM Figure 5 shows residues in 1y75A colored by their importance, at the interface with 1y75NAG301. Table 6. Residues forming surface ”patch” in 1y75A. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1y75A surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1y75. It is shown in Fig. 6. The right panel

4 Table 7. res type disruptive mutations 29 C (KE)(R)(QD)(MH) 39 D (R)(FWH)(Y)(VCAG) 27 C (FW)(R)(H)(YE) 51 C (E)(K)(R)(D) 26 G (KR)(E)(Q)(H) 30 G (R)(FW)(KH)(E) Fig. 7. Residues 1-120 in 1y75B colored by their relative importance. (See 45 C (E)(K)(R)(D) Appendix, Fig.12, for the coloring scheme.) 33 G (E)(R)(FKWH)(YD) 44 C (K)(R)(E)(Q) 48 H (E)(T)(MD)(VQA) Format: MSF 68 Y (K)(Q)(E)(M) Number of sequences: 518 22 F (KE)(T)(Q)(D) Total number of residues: 57320 35 G (R)(E)(KH)(FW) Smallest: 46 93 C (KR)(E)(Q)(H) Largest: 118 37 P (YR)(H)(TE)(K) Average length: 110.7 52 Y (K)(Q)(E)(M) Alignment length: 118 100 C (E)(K)(D)(R) Average identity: 44% 28 Y (K)(Q)(M)(ER) Most related pair: 99% 5 F (K)(TE)(QR)(CDG) Most unrelated pair: 0% 9 I (R)(Y)(T)(H) Most distant seq: 30% 49 C (R)(E)(FW)(KH) Furthermore, <1% of residues show as conserved in this ali- Table 7. Disruptive mutations for the surface patch in 1y75A. gnment. The alignment consists of 62% eukaryotic ( 61% vertebrata, <1% arthropoda, <1% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1y75B.descr. 3.3 Residue ranking in 1y75B The 1y75B sequence is shown in Fig. 7, with each residue colored according to its estimated importance. The full listing of residues in 1y75B can be found in the file called 1y75B.ranks sorted in the attachment. 3 CHAIN 1Y75B 3.4 Top ranking residues in 1y75B and their position on the structure 3.1 Q5G290 overview In the following we consider residues ranking among top 25% of From SwissProt, id Q5G290, 90% identical to 1y75B: residues in the protein . Figure 8 shows residues in 1y75B colored Description: Phospholipase A2 isoform 6 (Fragment). by their importance: bright red and yellow indicate more conser- Organism, scientific name: Naja sagittifera (Andaman cobra). ved/important residues (see Appendix for the coloring scheme). A Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Pymol script for producing this figure can be found in the attachment. Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; Colubroidea; Elapidae; Elapinae; Naja. Similarity: Belongs to the phospholipase A2 family. 3.4.1 Clustering of residues at 25% coverage. Fig. 9 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.9 are composed of the residues listed in Table 8. Table 8. 3.2 Multiple sequence alignment for 1y75B cluster size member For the chain 1y75B, the alignment 1y75B.msf (attached) with 518 color residues sequences was used. The alignment was downloaded from the HSSP red 27 5,22,25,26,27,28,29,30,33,35 database, and fragments shorter than 75% of the query as well as 37,39,41,42,44,45,48,49,51 duplicate sequences were removed. It can be found in the attachment continued in next column to this report, under the name of 1y75B.msf. Its statistics, from the alistat program are the following:

5 Table 8. Clusters of top ranking residues in 1y75B.

3.4.2 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 1y75A.Table 9 lists the top 25% of residues at the interface with 1y75A. The following table (Table 10) suggests possible disruptive replacements for these residues (see Section 4.6). Table 9. res type subst’s cvg noc/ dist (%) bb (A˚ ) 30 G G(97) 0.09 1/1 4.51 S(1).RD N

Table 9. The top 25% of residues in 1y75B at the interface with 1y75A. (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. 8. Residues in 1y75B, colored by their relative importance. Clockwise: front, back, top and bottom views. Table 10. res type disruptive mutations 30 G (R)(FW)(KE)(H)

Table 10. List of disruptive mutations for the top 25% of residues in 1y75B, that are at the interface with 1y75A.

Figure 10 shows residues in 1y75B colored by their importance, at the interface with 1y75A. 3.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1y75B surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1y75. It is shown in Fig. 11. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 11, while Table 12 suggests possible disruptive replacements for these residues (see Section 4.6). Table 11. res type substitutions(%) cvg antn 29 C C(99).W 0.01 S-S 27 C C(96)R.KN(1)E 0.03 S-S Fig. 9. Residues in 1y75B, colored according to the cluster they belong to: 39 D D(98).QEGP 0.03 red, followed by blue and yellow are the largest clusters (see Appendix for 51 C C(98).SF 0.04 S-S the coloring scheme). Clockwise: front, back, top and bottom views. The 26 G G(98).ASL 0.05 corresponding Pymol script is attached. 45 C C(98).YRL 0.07 S-S 33 G G(98).PNIKSA 0.08 30 G G(97)S(1).RDN 0.09 Table 8. continued 44 C C(98).AYWG 0.09 S-S 48 H H(98).RYQN 0.10 cluster size member color residues continued in next column 52,68,79,91,93,94,98,100 blue 2 61,86

6 Table 11. continued res type substitutions(%) cvg antn F(2)W(3).SE 49 D D(86)K(5)R.H 0.24 S(1)GN(2)EAQCM 9 I I(88)V(4).(1)MK 0.25 L(2)FNTSA

Table 11. Residues forming surface ”patch” in 1y75B.

Table 12. res type disruptive mutations 29 C (E)(K)(R)(D) 27 C (FW)(H)(E)(R) 39 D (R)(H)(FW)(Y) 51 C (K)(ER)(Q)(MD) 26 G (R)(K)(E)(H) 45 C (E)(K)(R)(D) 33 G (R)(E)(KH)(FYW) 30 G (R)(FW)(KE)(H) Fig. 10. Residues in 1y75B, at the interface with 1y75A, colored by their rela- 44 C (K)(E)(R)(Q) tive importance. 1y75A is shown in backbone representation (See Appendix 48 H (E)(T)(D)(M) for the coloring scheme for the protein chain 1y75B.) 68 Y (K)(Q)(E)(M) 37 P (YR)(H)(TE)(K) 93 C (KR)(E)(Q)(MHD) 22 F (KE)(T)(Q)(D) 35 G (R)(E)(FKWH)(Y) 100 C (E)(K)(D)(R) 52 Y (K)(Q)(E)(R) 5 F (K)(E)(TQ)(DR) 28 Y (K)(Q)(M)(ER) 49 D (R)(FW)(H)(Y) 9 I (Y)(R)(H)(T)

Table 12. Disruptive mutations for the surface patch in 1y75B. Fig. 11. A possible active surface on the chain 1y75B. The larger cluster it belongs to is shown in blue. 4 NOTES ON USING TRACE RESULTS Table 11. continued 4.1 Coverage res type substitutions(%) cvg antn Trace results are commonly expressed in terms of coverage: the resi- 68 Y Y(95)F.(1)CSLRI 0.13 due is important if its “coverage” is small - that is if it belongs to AN some small top percentage of residues [100% is all of the residues 37 P P(95).A(2)RTIGY 0.14 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 93 C C(96).(3)YL 0.14 S-S to some nearby percentage), sorted by the strength of the presumed 22 F Y(87)F(10).(1)L 0.15 evolutionary pressure. (I.e., the smaller the coverage, the stronger the N pressure on the residue.) Starting from the top of that list, mutating a 35 G G(93)KR(2).ENQH 0.16 couple of residues should affect the protein somehow, with the exact IPASD effects to be determined experimentally. 100 C C(95).(3)RYV 0.17 S-S 52 Y Y(94)CV.FLWHA 0.18 4.2 Known substitutions 5 F F(80)L(15).(3)Y 0.20 One of the table columns is “substitutions” - other amino acid types WE seen at the same position in the alignment. These amino acid types 28 Y H(4)N(7)Y(81) 0.21 may be interchangeable at that position in the protein, so if one wants continued in next column to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has

7 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, one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given COVERAGE in the cases when it is smaller than 1%. This is meant to be a rough

guide - due to rounding errors these percentages often do not add up V to 100%. 100% 50% 30% 5%

4.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by ˚ 2 at least 10A , which is roughly the area needed for one water mole- V cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor RELATIVE IMPORTANCE within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, Fig. 12. Coloring scheme used to color residues by their relative importance. 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- 5 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 5.1 File formats Files with extension “ranks sorted” are the actual trace results. The 4.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • alignment# number of the position in the alignment ber of contacts heavy atoms of the residue in question make across • residue# residue number in the PDB file the interface, as well as how many of them are realized through the • type amino acid type backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 1. number of different amino acids appearing in in this column 4.5 Annotation of the alignment If the residue annotation is available (either from the pdb file or 2. their type from other sources), another column, with the header “annotation” • rho ET score - the smaller this value, the lesser variability of appears. Annotations carried over from PDB are the following: site this position across the branches of the tree (and, presumably, (indicating existence of related site record in PDB ), S-S (disulfide the greater the importance for the protein) bond forming residue), hb (hydrogen bond forming residue, jb (james • cvg coverage - percentage of the residues on the structure which bond forming residue), and sb (for salt bridge forming residue). have this rho or smaller • gaps percentage of gaps in this column 4.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- 5.2 Color schemes used mentarity with the substitutions found in the alignment. Note that The following color scheme is used in figures with residues colored they are meant to be disruptive to the interaction of the protein by cluster size: black is a single-residue cluster; clusters composed of with its ligand. The attempt is made to complement the following more than one residue colored according to this hierarchy (ordered properties: small [AV GSTC], medium [LPNQDEMIK], large by descending size): red, blue, yellow, green, purple, azure, tur- [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tively [KHR], or negatively [DE] charged, aromatic [WFYH], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, long aliphatic chain [EKRQM], OH-group possession [SDETY ], DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, and NH2 group possession [NQRK]. The suggestions are listed tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. according to how different they appear to be from the original amino The colors used to distinguish the residues by the estimated acid, and they are grouped in round brackets if they appear equally evolutionary pressure they experience can be seen in Fig. 12. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 5.3 Credits disruptive) These suggestions are tentative - they might prove disrup- 5.3.1 Alistat alistat reads a multiple sequence alignment from the tive to the fold rather than to the interaction. Many researcher will file and shows a number of simple statistics about it. These stati- choose, however, the straightforward alanine mutations, especially in stics include the format, the number of sequences, the total number the beginning stages of their investigation. of residues, the average and range of the sequence lengths, and the

8 alignment length (e.g. including gap characters). Also shown are The viewer is self-unpacking and self-installing. Input files to be used some percent identities. A percent pairwise alignment identity is defi- with ETV (extension .etvx) can be found in the attachment to the ned as (idents / MIN(len1, len2)) where idents is the number of main report. exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and 5.5 Citing this work ”most unrelated pair” of the alignment are the average, maximum, The method used to rank residues and make predictions in this report and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of seq” is calculated by finding the maximum pairwise identity (best Evolution-Entropy Hybrid Methods for Ranking of Protein Residues relative) for all N sequences, then finding the minimum of these N by Importance” J. Mol. Bio. 336: 1265-82. For the original version numbers (hence, the most outlying sequence). alistat is copyrighted of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- by HHMI/Washington University School of Medicine, 1992-2001, tionary Trace Method Defines Binding Surfaces Common to Protein and freely distributed under the GNU General Public License. Families” J. Mol. Bio. 257: 342-358. report maker itself is described in Mihalek I., I. Res and O. 5.3.2 CE To map ligand binding sites from different Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type source structures, report maker uses the CE program: of service for comparative analysis of proteins.” Bioinformatics http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 22:1656-7. ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 5.6 About report maker 5.3.3 DSSP In this work a residue is considered solvent accessi- report maker was written in 2006 by Ivana Mihalek. The 1D ran- ble if the DSSP program finds it exposed to water by at least 10A˚ 2, king visualization program was written by Ivica Res.ˇ report maker which is roughly the area needed for one water molecule to come in is copyrighted by Lichtarge Lab, Baylor College of Medicine, the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Houston. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 5.7 Attachments by [email protected] November 18,2002, The following files should accompany this report: http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1y75A.complex.pdb - coordinates of 1y75A with all of its 5.3.4 HSSP Whenever available, report maker uses HSSP ali- interacting partners gnment as a starting point for the analysis (sequences shorter than • 1y75A.etvx - ET viewer input file for 1y75A 75% of the query are taken out, however); R. Schneider, A. de • 1y75A.cluster report.summary - Cluster report summary for Daruvar, and C. Sander. ”The HSSP database of protein structure- 1y75A sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 1y75A.ranks - Ranks file in sequence order for 1y75A http://swift.cmbi.kun.nl/swift/hssp/ • 1y75A.clusters - Cluster descriptions for 1y75A • 1y75A.msf - the multiple sequence alignment used for the chain 5.3.5 LaTex The text for this report was processed using LAT X; E 1y75A Leslie Lamport, “LaTeX: A Document Preparation System Addison- • Wesley,” Reading, Mass. (1986). 1y75A.descr - description of sequences used in 1y75A msf • 1y75A.ranks sorted - full listing of residues and their ranking 5.3.6 Muscle When making alignments “from scratch”, report for 1y75A maker uses Muscle alignment program: Edgar, Robert C. (2004), • ”MUSCLE: multiple sequence alignment with high accuracy and 1y75A.1y75B.if.pml - Pymol script for Figure 4 high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1y75A.cbcvg - used by other 1y75A – related pymol scripts • 1y75A.1y75NAG301.if.pml - Pymol script for Figure 5 http://www.drive5.com/muscle/ • 1y75B.complex.pdb - coordinates of 1y75B with all of its 5.3.7 Pymol The figures in this report were produced using interacting partners Pymol. The scripts can be found in the attachment. Pymol • 1y75B.etvx - ET viewer input file for 1y75B is an open-source application copyrighted by DeLano Scien- • 1y75B.cluster report.summary - Cluster report summary for tific LLC (2005). For more information about Pymol see 1y75B http://pymol.sourceforge.net/. (Note for Windows • 1y75B.ranks - Ranks file in sequence order for 1y75B users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) • 1y75B.clusters - Cluster descriptions for 1y75B • 1y75B.msf - the multiple sequence alignment used for the chain 5.4 Note about ET Viewer 1y75B Dan Morgan from the Lichtarge lab has developed a visualization • 1y75B.descr - description of sequences used in 1y75B msf tool specifically for viewing trace results. If you are interested, please • 1y75B.ranks sorted - full listing of residues and their ranking visit: for 1y75B http://mammoth.bcm.tmc.edu/traceview/ • 1y75B.1y75A.if.pml - Pymol script for Figure 10

9 • 1y75B.cbcvg - used by other 1y75B – related pymol scripts

10