Pages 1–8 3c1o Evolutionary trace report by report maker January 4, 2010 4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 8 4.4 Note about ET Viewer 8 4.5 Citing this work 8 4.6 About report maker 8 4.7 Attachments 8 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3c1o): Title: The multiple phenylpropene synthases in both clarkia breweri and petunia hybrida represent two distinct lineages CONTENTS Compound: Mol id: 1; molecule: eugenol synthase; chain: a; synonym: brewer’s clarkia; engineered: yes 1 Introduction 1 Organism, scientific name: Clarkia Breweri; 3c1o contains a single unique chain 3c1oA (314 residues long). 2 Chain 3c1oA 1 2.1 O49820 overview 1 2.2 Multiple sequence alignment for 3c1oA 1 2.3 Residue ranking in 3c1oA 1 2.4 Top ranking residues in 3c1oA and their position on the structure 2 2 CHAIN 3C1OA 2.4.1 Clustering of residues at 25% coverage. 2 2.1 O49820 overview 2.4.2 Overlap with known functional surfaces at 25% coverage. 2 From SwissProt, id O49820, 57% identical to 3c1oA: 2.4.3 Possible novel functional surfaces at 25% Description: Isoflavone reductase-like protein. coverage. 3 Organism, scientific name: Citrus paradisi (Grapefruit). Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 3 Notes on using trace results 6 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3.1 Coverage 6 eudicots; rosids; eurosids II; Sapindales; Rutaceae; Citrus. 3.2 Known substitutions 6 3.3 Surface 6 3.4 Number of contacts 6 3.5 Annotation 6 2.2 Multiple sequence alignment for 3c1oA 3.6 Mutation suggestions 7 For the chain 3c1oA, the alignment 3c1oA.msf (attached) with 134 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 7 database, and fragments shorter than 75% of the query as well as 4.1 File formats 7 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 7 to this report, under the name of 3c1oA.msf. Its statistics, from the 4.3 Credits 7 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 1-157 in 3c1oA colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.) Fig. 2. Residues 158-314 in 3c1oA colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.) Fig. 3. Residues in 3c1oA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 134 Total number of residues: 40367 Smallest: 239 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 314 top 25% of all residues, this time colored according to clusters they Average length: 301.2 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 314 in Table 1. Average identity: 48% Most related pair: 99% Table 1. Most unrelated pair: 23% cluster size member Most distant seq: 46% color residues red 57 5,20,21,28,31,33,54,55,60,64 68,75,76,78,79,80,90,93,96 Furthermore, <1% of residues show as conserved in this ali- 97,98,100,101,102,104,105 gnment. 106,108,109,110,111,112,114 The alignment consists of 35% eukaryotic ( 35% plantae) 115,123,124,128,131,135,139 sequences. (Descriptions of some sequences were not readily availa- 144,146,189,191,193,196,202 ble.) The file containing the sequence descriptions can be found in 207,208,211,214,217,284,288 the attachment, under the name 3c1oA.descr. 289,290,294 blue 6 170,228,229,231,236,238 2.3 Residue ranking in 3c1oA yellow 4 176,178,179,180 The 3c1oA sequence is shown in Figs. 1–2, with each residue colored green 4 8,10,11,14 according to its estimated importance. The full listing of residues purple 2 247,265 in 3c1oA can be found in the file called 3c1oA.ranks sorted in the attachment. Table 1. Clusters of top ranking residues in 3c1oA. 2.4 Top ranking residues in 3c1oA 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 3c1oA colored and the heteroatom name used in that file. 2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) S(2) Y(2) A(2)G .(2) 151 N YN(85) 0.25 8/8 2.78 site H(5) G(2)S .(2) A(1)F Table 2. The top 25% of residues in 3c1oA at the interface with NAP.(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 3. Fig. 4. Residues in 3c1oA, colored according to the cluster they belong to: res type disruptive red, followed by blue and yellow are the largest clusters (see Appendix for mutations the coloring scheme). Clockwise: front, back, top and bottom views. The 8 G (KER)(FQMWHD)(NLPI)(Y) corresponding Pymol script is attached. 11 G (KER)(FQMWHD)(NLPI)(Y) 14 G (KER)(FQMWHD)(NLPI)(Y) 111 F (K)(E)(Q)(D) NAP binding site. Table 2 lists the top 25% of residues at the 131 K (FYW)(VA)(H)(T) interface with 3c1oANAP401 (nap). The following table (Table 3) 33 R (T)(D)(Y)(SCG) suggests possible disruptive replacements for these residues (see 10 T (R)(K)(H)(FW) Section 3.6). 112 G (KER)(FWH)(D)(M) 109 S (K)(R)(FQMWH)(YE) Table 2. 110 D (R)(H)(FW)(K) res type subst’s cvg noc/ dist antn 157 F (K)(E)(Q)(R) (%) bb (A˚ ) 151 N (Y)(E)(R)(TH) 8 G G(99). 0.03 15/15 3.58 site 11 G G(99). 0.03 20/20 2.95 site 14 G G(99). 0.03 1/1 4.43 Table 3. List of disruptive mutations for the top 25% of residues in 3c1oA, that are at the interface with NAP. 111 F .F(94) 0.06 16/10 3.00 Y(5) 131 K K(97)S. 0.06 10/0 2.83 site Figure 5 shows residues in 3c1oA colored by their importance, at the TE interface with 3c1oANAP401. 33 R R(97)XM 0.07 77/1 2.83 site Interface with 3c1oA1.Table 4 lists the top 25% of residues at . the interface with 3c1oA1. The following table (Table 5) suggests 10 T T(95) 0.09 29/12 2.58 site possible disruptive replacements for these residues (see Section 3.6). S(2)M. Table 4. 112 G .G(97)E 0.10 6/6 3.71 res type subst’s cvg noc/ dist antn AR (%) bb (A˚ ) 109 S .S(95) 0.12 14/5 2.71 site 33 R R(97)XM 0.07 1/0 4.77 site A(2)R . 110 D .E(92) 0.15 10/10 3.33 site D(5)AS 157 F L(6) 0.23 6/0 3.12 site Table 4. The top 25% of residues in 3c1oA at the interface with 3c1oA1. F(61) (Field names: res: residue number in the PDB entry; type: amino acid type; P(20) 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 continued in next column contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) 3 Table 5. res type disruptive mutations 33 R (T)(D)(Y)(SCG) Table 5. List of disruptive mutations for the top 25% of residues in 3c1oA, that are at the interface with 3c1oA1. Fig. 5. Residues in 3c1oA, at the interface with NAP, colored by their relative importance. The ligand (NAP) 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 3c1oA.) Fig. 6. Residues in 3c1oA, at the interface with 3c1oA1, colored by their rela- tive importance. 3c1oA1 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 3c1oA.) Figure 6 shows residues in 3c1oA colored by their importance, at the interface with 3c1oA1. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 3c1oA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 3c1o. It is shown in Fig. 7. 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 6, while Table 7 suggests possible disruptive replacements for these residues (see Section 3.6).