Pages 1–10 1mh2 Evolutionary trace report by report maker July 21, 2010

4 Notes on using trace results 7 4.1 Coverage 7 4.2 Known substitutions 8 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 9 5.3 Credits 9 5.3.1 Alistat 9 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 5.6 About report maker 9 CONTENTS 5.7 Attachments 9

1 Introduction 1 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1mh2): 2 Chain 1mh2A 1 Title: Crystal structure of a zinc containing dimer of phospholipase 2.1 P60043 overview 1 a2 from the venom of indian cobra (naja naja sagittifera) 2.2 Multiple sequence alignment for 1mh2A 1 Compound: Mol id: 1; molecule: phospholipase a2; chain: a; ec: 2.3 Residue ranking in 1mh2A 1 3.1.1.4; mol id: 2; molecule: phospholipase a2; chain: b; ec: 3.1.1.4 2.4 Top ranking residues in 1mh2A and their position on Organism, scientific name: Naja Sagittifera; the structure 1 1mh2 contains unique chains 1mh2A (119 residues) and 1mh2B 2.4.1 Clustering of residues at 25% coverage. 2 (119 residues) 2.4.2 Overlap with known functional surfaces at 25% coverage. 2 2 CHAIN 1MH2A 2.4.3 Possible novel functional surfaces at 25% coverage. 3 2.1 P60043 overview From SwissProt, id P60043, 93% identical to 1mh2A: 3 Chain 1mh2B 4 Description: Phospholipase A2 isoform 1 precursor (EC 3.1.1.4) 3.1 P60044 overview 4 (Phosphatidylcholine 2-acylhydrolase) (Fragment). 3.2 Multiple sequence alignment for 1mh2B 5 Organism, scientific name: Naja sagittifera (Andaman cobra). 3.3 Residue ranking in 1mh2B 5 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.4 Top ranking residues in 1mh2B and their position on Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; the structure 5 Colubroidea; Elapidae; Elapinae; Naja. 3.4.1 Clustering of residues at 25% coverage. 5 Function: PA2 catalyzes the calcium-dependent hydrolysis of the 2- 3.4.2 Overlap with known functional surfaces at acyl groups in 3-sn-phosphoglycerides. 25% coverage. 6 Catalytic activity: Phosphatidylcholine + H(2)O = 1- acylglycero- 3.4.3 Possible novel functional surfaces at 25% phosphocholine + a carboxylate. coverage. 6 Cofactor: Binds 1 calcium ion per subunit (By similarity).

1 Lichtarge lab 2006 2.4 Top ranking residues in 1mh2A and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 1mh2A colored 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 1-120 in 1mh2A colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.)

Subunit: Heterodimer formed between two homologous isoforms: isoform 1 and isoform 2. Subcellular location: Secreted. Similarity: Belongs to the phospholipase A2 family. Group I subfa- mily. About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are no restrictions on its use as long as its content is in no way modified and this statement is not removed.

2.2 Multiple sequence alignment for 1mh2A For the chain 1mh2A, the alignment 1mh2A.msf (attached) with 538 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 1mh2A.msf. Its statistics, from the alistat program are the following: Fig. 2. Residues in 1mh2A, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 538 Total number of residues: 58778 Smallest: 42 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Largest: 119 top 25% of all residues, this time colored according to clusters they Average length: 109.3 belong to. The clusters in Fig.3 are composed of the residues listed Alignment length: 119 in Table 1. Average identity: 43% Most related pair: 99% Table 1. Most unrelated pair: 0% cluster size member Most distant seq: 32% color residues red 27 2,5,8,9,22,25,26,27,28,29,30 35,37,39,41,42,44,45,48,51 Furthermore, <1% of residues show as conserved in this ali- 52,68,79,91,93,94,100 gnment. blue 2 61,86 The alignment consists of 61% eukaryotic ( 59% vertebrata, <1% arthropoda, <1% plantae), and <1% prokaryotic sequences. (Des- Table 1. Clusters of top ranking residues in 1mh2A. criptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1mh2A.descr. 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier 2.3 Residue ranking in 1mh2A and the heteroatom name used in that file. Interface with 1mh2B.Table 2 lists the top 25% of residues at The 1mh2A sequence is shown in Fig. 1, with each residue colored the interface with 1mh2B. The following table (Table 3) suggests according to its estimated importance. The full listing of residues in possible disruptive replacements for these residues (see Section 4.6). 1mh2A can be found in the file called 1mh2A.ranks sorted in the attachment.

2 Fig. 3. Residues in 1mh2A, colored according to the cluster they belong to: Fig. 4. Residues in 1mh2A, at the interface with 1mh2B, colored by their rela- red, followed by blue and yellow are the largest clusters (see Appendix for tive importance. 1mh2B is shown in backbone representation (See Appendix the coloring scheme). Clockwise: front, back, top and bottom views. The for the coloring scheme for the protein chain 1mh2A.) corresponding Pymol script is attached.

Acetic acid binding site. Table 4 lists the top 25% of residues Table 2. at the interface with 1mh2ACY302 (acetic acid). The following table res type subst’s cvg noc/ dist (Table 5) suggests possible disruptive replacements for these residues (%) bb (A˚ ) (see Section 4.6). 30 G G(96)NL 0.06 2/2 4.25 Table 4. S(1).VT res type subst’s cvg noc/ dist antn RD (%) bb (A˚ ) 32 G G(92)S. 0.10 18/18 3.18 30 G G(96)NL 0.06 1/1 4.89 L(2) S(1).VT E(1)VAN RD KQIRM 45 C C(96)SE 0.09 1/1 4.32 S-S K.DAYRQ Table 2. The top 25% of residues in 1mh2A at the interface with 1mh2B. 48 H H(95)PQ 0.13 16/4 2.48 (Field names: res: residue number in the PDB entry; type: amino acid type; .LRYSVT substs: substitutions seen in the alignment; with the percentage of each type INGE in the bracket; noc/bb: number of contacts with the ligand, with the number of 52 Y Y(91)DT 0.22 6/0 4.42 contacts realized through backbone atoms given in the bracket; dist: distance CV(2) of closest apporach to the ligand. ) F(1) .(1)HLS WIP Table 3. res type disruptive Table 4. The top 25% of residues in 1mh2A at the interface with acetic mutations acid.(Field names: res: residue number in the PDB entry; type: amino acid 30 G (R)(K)(E)(H) type; substs: substitutions seen in the alignment; with the percentage of each 32 G (R)(EH)(Y)(FKW) 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 3. List of disruptive mutations for the top 25% of residues in 1mh2A, that are at the interface with 1mh2B.

Figure 4 shows residues in 1mh2A colored by their importance, at the interface with 1mh2B.

3 Table 5. res type disruptive mutations 30 G (R)(K)(E)(H) 45 C (R)(FKW)(EH)(M) 48 H (E)(TD)(Q)(M) 52 Y (K)(Q)(R)(M)

Table 5. List of disruptive mutations for the top 25% of residues in 1mh2A, that are at the interface with acetic acid.

Fig. 6. A possible active surface on the chain 1mh2A. The larger cluster it belongs to is shown in blue.

Table 6. continued res type substitutions(%) cvg antn 51 C C(98).PF 0.03 S-S 35 G G(95)Q.AR(1) 0.04 H(1)EPND 44 C C(97).AVPYWG 0.05 S-S 30 G G(96)NLS(1).VTR 0.06 D 26 G G(95)TR.DSALCN 0.08 39 D D(96)T.(1)KVENG 0.08 45 C C(96)SEK.DAYRQ 0.09 S-S 32 G G(92)S.L(2)E(1) 0.10 VANKQIRM 48 H H(95)PQ.LRYSVTI 0.13 NGE 68 Y Y(93)FR.(2)CSIA 0.13 LM 5 F F(77).(7)L(14)M 0.14 YIE 100 C C(92).(6)NGRYK 0.15 S-S Fig. 5. Residues in 1mh2A, at the interface with acetic acid, colored by their 22 F Y(83)I.(3)F(11) 0.18 relative importance. The ligand (acetic acid) is colored green. Atoms further NLVT 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 37 P P(93)Q.(1)EG 0.18 for the protein chain 1mh2A.) A(2)TDILCYS 93 C C(93).(4)NSYLA 0.19 S-S 9 I I(85)T.(5)V(4)M 0.21 Figure 5 shows residues in 1mh2A colored by their importance, at NL(1)FSA the interface with 1mh2ACY302. 52 Y Y(91)DTCV(2) 0.22 F(1).(1)HLSWIP 2.4.3 Possible novel functional surfaces at 25% coverage. One 28 Y H(4)TN(7)Y(78) 0.23 group of residues is conserved on the 1mh2A surface, away from (or F(2)W(3)P.LARDS susbtantially larger than) other functional sites and interfaces reco- E gnizable in PDB entry 1mh2. It is shown in Fig. 6. The right panel 2 T .(20)L(71)V(3)Q 0.24 shows (in blue) the rest of the larger cluster this surface belongs to. I(1)MATR The residues belonging to this surface ”patch” are listed in Table 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 4.6). Table 6. Residues forming surface ”patch” in 1mh2A. Table 6. res type substitutions(%) cvg antn 27 C C(95)RK(1).N(2) 0.02 S-S LSM continued in next column

4 Table 7. res type disruptive mutations 27 C (E)(R)(FWH)(Y) 51 C (KER)(QD)(H)(M) 35 G (R)(E)(K)(FWH) 44 C (K)(ER)(Q)(D) 30 G (R)(K)(E)(H) 26 G (R)(K)(E)(H) Fig. 7. Residues 1-120 in 1mh2B colored by their relative importance. (See 39 D (R)(FWH)(Y)(K) Appendix, Fig.12, for the coloring scheme.) 45 C (R)(FKW)(EH)(M) 32 G (R)(EH)(Y)(FKW) 48 H (E)(TD)(Q)(M) to this report, under the name of 1mh2B.msf. Its statistics, from the 68 Y (K)(Q)(R)(E) alistat program are the following: 5 F (K)(T)(E)(R) 100 C (E)(FW)(KR)(D) Format: MSF 22 F (K)(E)(QR)(D) Number of sequences: 538 37 P (R)(Y)(H)(K) Total number of residues: 58321 93 C (R)(K)(E)(H) Smallest: 32 9 I (R)(Y)(H)(K) Largest: 119 52 Y (K)(Q)(R)(M) Average length: 108.4 28 Y (K)(Q)(R)(M) Alignment length: 119 2 T (R)(H)(K)(FW) Average identity: 44% Most related pair: 99% Table 7. Disruptive mutations for the surface patch in 1mh2A. Most unrelated pair: 0% Most distant seq: 30%

Furthermore, <1% of residues show as conserved in this ali- 3 CHAIN 1MH2B gnment. 3.1 P60044 overview The alignment consists of 61% eukaryotic ( 59% vertebrata, <1% From SwissProt, id P60044, 94% identical to 1mh2B: arthropoda, <1% plantae), and <1% prokaryotic sequences. (Des- Description: Phospholipase A2 isoform 2 precursor (EC 3.1.1.4) criptions of some sequences were not readily available.) The file (Phosphatidylcholine 2-acylhydrolase) (Fragment). containing the sequence descriptions can be found in the attachment, Organism, scientific name: Naja sagittifera (Andaman cobra). under the name 1mh2B.descr. Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.3 Residue ranking in 1mh2B Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; Colubroidea; Elapidae; Elapinae; Naja. The 1mh2B sequence is shown in Fig. 7, with each residue colored Function: PA2 catalyzes the calcium-dependent hydrolysis of the 2- according to its estimated importance. The full listing of residues acyl groups in 3-sn-phosphoglycerides. in 1mh2B can be found in the file called 1mh2B.ranks sorted in the Catalytic activity: Phosphatidylcholine + H(2)O = 1- acylglycero- attachment. phosphocholine + a carboxylate. 3.4 Top ranking residues in 1mh2B and their position on Cofactor: Binds 1 calcium ion per subunit (By similarity). the structure Subunit: Heterodimer formed between two homologous isoforms: isoform 1 and isoform 2. In the following we consider residues ranking among top 25% of Subcellular location: Secreted. residues in the protein . Figure 8 shows residues in 1mh2B colored Similarity: Belongs to the phospholipase A2 family. Group I subfa- by their importance: bright red and yellow indicate more conser- mily. ved/important residues (see Appendix for the coloring scheme). A About: This Swiss-Prot entry is copyright. It is produced through a Pymol script for producing this figure can be found in the attachment. collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are 3.4.1 Clustering of residues at 25% coverage. Fig. 9 shows the no restrictions on its use as long as its content is in no way modified top 25% of all residues, this time colored according to clusters they and this statement is not removed. belong to. The clusters in Fig.9 are composed of the residues listed in Table 8. 3.2 Multiple sequence alignment for 1mh2B For the chain 1mh2B, the alignment 1mh2B.msf (attached) with 538 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

5 Table 8. continued cluster size member color residues red 28 5,9,22,25,26,27,28,29,30,32 33,35,37,39,41,42,44,45,48 49,51,52,68,79,91,93,94,100 blue 2 61,86

Table 8. Clusters of top ranking residues in 1mh2B.

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 1mh2A.Table 9 lists the top 25% of residues at the interface with 1mh2A. 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 antn (%) bb (A˚ ) 29 C C(99)P. 0.01 8/8 2.71 S-S Fig. 8. Residues in 1mh2B, colored by their relative importance. Clockwise: W front, back, top and bottom views. 27 C C(95)R 0.02 8/8 3.89 S-S K(1). N(2)SM 25 Y Y(97).H 0.03 3/3 4.48 CAGQ 30 G G(96)NP 0.07 4/4 4.56 S(1).VK DR 26 G G(96)T. 0.09 3/3 3.66 RAISNLC 32 G G(92)QS 0.11 9/9 2.72 L(2). E(1)VAN KIRM 33 G G(87) 0.22 1/1 4.96 R(1) W(1)H.Y N(1) S(2)QTE FVPKDA 28 Y H(4)T 0.23 2/2 4.68 N(7) Y(78) F(2) W(3)P.A Fig. 9. Residues in 1mh2B, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for SDMRE the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 9. The top 25% of residues in 1mh2B at the interface with 1mh2A. (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 Table 8. in the bracket; noc/bb: number of contacts with the ligand, with the number of cluster size member contacts realized through backbone atoms given in the bracket; dist: distance color residues of closest apporach to the ligand. ) continued in next column

6 Table 10. res type disruptive mutations 29 C (KER)(QD)(H)(M) 27 C (E)(FW)(HR)(YD) 25 Y (K)(QM)(E)(R) 30 G (R)(E)(FKWH)(Y) 26 G (R)(E)(K)(H) 32 G (R)(EH)(Y)(FKW) 33 G (R)(KE)(H)(D) 28 Y (K)(Q)(R)(M)

Fig. 11. A possible active surface on the chain 1mh2B. The larger cluster it Table 10. List of disruptive mutations for the top 25% of residues in belongs to is shown in blue. 1mh2B, that are at the interface with 1mh2A.

Table 11. res type substitutions(%) cvg antn 29 C C(99)P.W 0.01 S-S 27 C C(95)RK(1).N(2) 0.02 S-S SM 39 D D(97)TEK.ANF 0.03 51 C C(98).F 0.04 S-S 45 C C(97)SMK.HYRETQ 0.05 S-S 44 C C(97).AVPRYWG 0.06 S-S 30 G G(96)NPS(1).VKD 0.07 R 35 G G(94)QR(1).A 0.08 H(1)EPCND 26 G G(96)T.RAISNLC 0.09 48 H H(96)P.RYQVNGE 0.10 32 G G(92)QSL(2). 0.11 E(1)VANKIRM 5 F F(77).(7)L(14)M 0.12 IYE 37 P P(94)QYE.A(2)NT 0.13 DIGLS 68 Y Y(93)FLV.(2)C 0.14 S(1)RIAK 100 C C(93).(5)ENRYSD 0.16 S-S Fig. 10. Residues in 1mh2B, at the interface with 1mh2A, colored by 22 F Y(84)I.(3)F(10) 0.18 their relative importance. 1mh2A is shown in backbone representation (See L(1)VN Appendix for the coloring scheme for the protein chain 1mh2B.) 93 C C(93).(4)PMNDYE 0.18 S-S LW Figure 10 shows residues in 1mh2B colored by their importance, at 9 I I(86)T.(5)V(4)M 0.19 the interface with 1mh2A. KL(1)FNWSA 52 Y Y(92)DLCV(1)F 0.21 3.4.3 Possible novel functional surfaces at 25% coverage. One .(1)NWIHP group of residues is conserved on the 1mh2B surface, away from (or 33 G G(87)R(1)W(1)H. 0.22 susbtantially larger than) other functional sites and interfaces reco- YN(1)S(2)QTEFVP gnizable in PDB entry 1mh2. It is shown in Fig. 11. The right panel KDA shows (in blue) the rest of the larger cluster this surface belongs to. 28 Y H(4)TN(7)Y(78) 0.23 The residues belonging to this surface ”patch” are listed in Table 11, F(2)W(3)P.ASDMR while Table 12 suggests possible disruptive replacements for these continued in next column residues (see Section 4.6).

7 Table 11. continued an R at that position, it is advisable to try anything, but RVK. Conver- res type substitutions(%) cvg antn sely, when looking for substitutions which will not affect the protein, E one may try replacing, R with K, or (perhaps more surprisingly), with 49 D D(85)RK(5)N(2). 0.24 V. The percentage of times the substitution appears in the alignment HS(1)EGACQM 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 Table 11. Residues forming surface ”patch” in 1mh2B. guide - due to rounding errors these percentages often do not add up to 100%.

Table 12. 4.3 Surface res type disruptive To detect candidates for novel functional interfaces, first we look for mutations residues that are solvent accessible (according to DSSP program) by 2 29 C (KER)(QD)(H)(M) at least 10A˚ , which is roughly the area needed for one water mole- 27 C (E)(FW)(HR)(YD) cule to come in the contact with the residue. Furthermore, we require 39 D (R)(H)(FW)(Y) that these residues form a “cluster” of residues which have neighbor 51 C (E)(K)(R)(D) within 5A˚ from any of their heavy atoms. 45 C (ER)(K)(FW)(H) Note, however, that, if our picture of protein evolution is correct, 44 C (E)(K)(R)(D) the neighboring residues which are not surface accessible might be 30 G (R)(E)(FKWH)(Y) equally important in maintaining the interaction specificity - they 35 G (R)(E)(K)(FWH) should not be automatically dropped from consideration when choo- 26 G (R)(E)(K)(H) sing the set for mutagenesis. (Especially if they form a cluster with 48 H (E)(T)(D)(M) the surface residues.) 32 G (R)(EH)(Y)(FKW) 5 F (K)(T)(E)(R) 4.4 Number of contacts 37 P (R)(Y)(H)(TK) 68 Y (K)(Q)(E)(M) Another column worth noting is denoted “noc/bb”; it tells the num- 100 C (R)(K)(FW)(EH) ber of contacts heavy atoms of the residue in question make across 22 F (KE)(T)(QR)(D) the interface, as well as how many of them are realized through the 93 C (R)(K)(H)(E) backbone atoms (if all or most contacts are through the backbone, 9 I (YR)(H)(T)(E) mutation presumably won’t have strong impact). Two heavy atoms ˚ 52 Y (K)(Q)(R)(E) are considered to be “in contact” if their centers are closer than 5A. 33 G (R)(KE)(H)(D) 28 Y (K)(Q)(R)(M) 4.5 Annotation 49 D (R)(FW)(H)(Y) If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” Table 12. Disruptive mutations for the surface patch in 1mh2B. 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). 4 NOTES ON USING TRACE RESULTS 4.1 Coverage 4.6 Mutation suggestions Trace results are commonly expressed in terms of coverage: the resi- Mutation suggestions are completely heuristic and based on comple- due is important if its “coverage” is small - that is if it belongs to mentarity with the substitutions found in the alignment. Note that some small top percentage of residues [100% is all of the residues they are meant to be disruptive to the interaction of the protein in a chain], according to trace. The ET results are presented in the with its ligand. The attempt is made to complement the following form of a table, usually limited to top 25% percent of residues (or properties: small [AV GSTC], medium [LPNQDEMIK], large to some nearby percentage), sorted by the strength of the presumed [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- evolutionary pressure. (I.e., the smaller the coverage, the stronger the tively [KHR], or negatively [DE] charged, aromatic [WFYH], pressure on the residue.) Starting from the top of that list, mutating a long aliphatic chain [EKRQM], OH-group possession [SDETY ], couple of residues should affect the protein somehow, with the exact and NH2 group possession [NQRK]. The suggestions are listed effects to be determined experimentally. according to how different they appear to be from the original amino acid, and they are grouped in round brackets if they appear equally 4.2 Known substitutions disruptive. From left to right, each bracketed group of amino acid One of the table columns is “substitutions” - other amino acid types types resembles more strongly the original (i.e. is, presumably, less seen at the same position in the alignment. These amino acid types disruptive) These suggestions are tentative - they might prove disrup- may be interchangeable at that position in the protein, so if one wants tive to the fold rather than to the interaction. Many researcher will to affect the protein by a point mutation, they should be avoided. For choose, however, the straightforward alanine mutations, especially in example if the substitutions are “RVK” and the original protein has the beginning stages of their investigation.

8 alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two COVERAGE sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum,

V and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant 100% 50% 30% 5% seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License.

V 5.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: RELATIVE IMPORTANCE http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension Fig. 12. Coloring scheme used to color residues by their relative importance. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 5.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 5 APPENDIX 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 5.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: by [email protected] November 18,2002,

• alignment# number of the position in the alignment http://www.cmbi.kun.nl/gv/dssp/descrip.html. • residue# residue number in the PDB file 5.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type gnment as a starting point for the analysis (sequences shorter than • rank rank of the position according to older version of ET 75% of the query are taken out, however); R. Schneider, A. de • variability has two subfields: Daruvar, and C. Sander. ”The HSSP database of protein structure- 1. number of different amino acids appearing in in this column sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of the alignment http://swift.cmbi.kun.nl/swift/hssp/ 2. their type • rho ET score - the smaller this value, the lesser variability of 5.3.5 LaTex The text for this report was processed using LATEX; this position across the branches of the tree (and, presumably, Leslie Lamport, “LaTeX: A Document Preparation System Addison- the greater the importance for the protein) Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which 5.3.6 Muscle When making alignments “from scratch”, report have this rho or smaller maker uses Muscle alignment program: Edgar, Robert C. (2004), • gaps percentage of gaps in this column ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. 5.2 Color schemes used The following color scheme is used in figures with residues colored http://www.drive5.com/muscle/ by cluster size: black is a single-residue cluster; clusters composed of more than one residue colored according to this hierarchy (ordered 5.3.7 Pymol The figures in this report were produced using by descending size): red, blue, yellow, green, purple, azure, tur- Pymol. The scripts can be found in the attachment. Pymol quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, is an open-source application copyrighted by DeLano Scien- bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, tific LLC (2005). For more information about Pymol see DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, http://pymol.sourceforge.net/. (Note for Windows tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. users: the attached package needs to be unzipped for Pymol to read The colors used to distinguish the residues by the estimated the scripts and launch the viewer.) evolutionary pressure they experience can be seen in Fig. 12. 5.4 Note about ET Viewer 5.3 Credits Dan Morgan from the Lichtarge lab has developed a visualization 5.3.1 Alistat alistat reads a multiple sequence alignment from the tool specifically for viewing trace results. If you are interested, please file and shows a number of simple statistics about it. These stati- visit: stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the http://mammoth.bcm.tmc.edu/traceview/

9 The viewer is self-unpacking and self-installing. Input files to be used • 1mh2A.ranks - Ranks file in sequence order for 1mh2A with ETV (extension .etvx) can be found in the attachment to the • 1mh2A.clusters - Cluster descriptions for 1mh2A main report. • 1mh2A.msf - the multiple sequence alignment used for the chain 5.5 Citing this work 1mh2A The method used to rank residues and make predictions in this report • 1mh2A.descr - description of sequences used in 1mh2A msf can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1mh2A.ranks sorted - full listing of residues and their ranking Evolution-Entropy Hybrid Methods for Ranking of Protein Residues for 1mh2A by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1mh2A.1mh2B.if.pml - Pymol script for Figure 4 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- tionary Trace Method Defines Binding Surfaces Common to Protein • 1mh2A.cbcvg - used by other 1mh2A – related pymol scripts Families” J. Mol. Bio. 257: 342-358. • 1mh2A.1mh2ACY302.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 1mh2B.complex.pdb - coordinates of 1mh2B with all of its Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type interacting partners of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 1mh2B.etvx - ET viewer input file for 1mh2B • 1mh2B.cluster report.summary - Cluster report summary for 5.6 About report maker 1mh2B report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 1mh2B.ranks - Ranks file in sequence order for 1mh2B king visualization program was written by Ivica Res.ˇ report maker • is copyrighted by Lichtarge Lab, Baylor College of Medicine, 1mh2B.clusters - Cluster descriptions for 1mh2B Houston. • 1mh2B.msf - the multiple sequence alignment used for the chain 1mh2B 5.7 Attachments • 1mh2B.descr - description of sequences used in 1mh2B msf The following files should accompany this report: • 1mh2B.ranks sorted - full listing of residues and their ranking • 1mh2A.complex.pdb - coordinates of 1mh2A with all of its for 1mh2B interacting partners • 1mh2B.1mh2A.if.pml - Pymol script for Figure 10 • 1mh2A.etvx - ET viewer input file for 1mh2A • 1mh2B.cbcvg - used by other 1mh2B – related pymol scripts • 1mh2A.cluster report.summary - Cluster report summary for 1mh2A

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