Pages 1–6 1hro Evolutionary trace report by report maker September 28, 2008 4.3.1 Alistat 5 4.3.2 CE 6 4.3.3 DSSP 6 4.3.4 HSSP 6 4.3.5 LaTex 6 4.3.6 Muscle 6 4.3.7 Pymol 6 4.4 Note about ET Viewer 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 6 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1hro): Title: Molecular structure of a high potential cytochrome c2 isolated from rhodopila globiformis Compound: Mol id: 1; molecule: cytochrome c2; chain: a, b Organism, scientific name: Rhodopila Globiformis 1hro contains a single unique chain 1hroA (105 residues long) and its homologue 1hroB. CONTENTS 1 Introduction 1 2 CHAIN 1HROA 2.1 P00080 overview 2 Chain 1hroA 1 From SwissProt, id P00080, 98% identical to 1hroA: 2.1 P00080 overview 1 Description: Cytochrome c2. 2.2 Multiple sequence alignment for 1hroA 1 Organism, scientific name: Rhodopila globiformis (Rhodopseudo- 2.3 Residue ranking in 1hroA 1 monas globiformis). 2.4 Top ranking residues in 1hroA and their position on Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Rhodo- the structure 1 spirillales; Acetobacteraceae; Rhodopila. 2.4.1 Clustering of residues at 25% coverage. 2 Biophysicochemical properties: 2.4.2 Overlap with known functional surfaces at Redox potential: E(0) is +450 mV; 25% coverage. 2 Ptm: Binds 1 heme group per subunit. 2.4.3 Possible novel functional surfaces at 25% Similarity: Belongs to the cytochrome c family. coverage. 3 About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 4 collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are 3.1 Coverage 4 no restrictions on its use as long as its content is in no way modified 3.2 Known substitutions 4 and this statement is not removed. 3.3 Surface 5 3.4 Number of contacts 5 2.2 Multiple sequence alignment for 1hroA 3.5 Annotation 5 3.6 Mutation suggestions 5 For the chain 1hroA, the alignment 1hroA.msf (attached) with 31 sequences was used. The alignment was assembled through combi- 4 Appendix 5 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 5 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 5 report, under the name of 1hroA.msf. Its statistics, from the alistat 4.3 Credits 5 program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 2-106 in 1hroA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Format: MSF Number of sequences: 31 Total number of residues: 3204 Smallest: 101 Largest: 105 Average length: 103.4 Alignment length: 105 Average identity: 48% Most related pair: 98% Most unrelated pair: 30% Most distant seq: 51% Fig. 2. Residues in 1hroA, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, 11% of residues show as conserved in this alignment. The alignment consists of 41% eukaryotic ( 22% vertebrata, 12% plantae), and 54% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1hroA.descr. 2.3 Residue ranking in 1hroA The 1hroA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1hroA can be found in the file called 1hroA.ranks sorted in the attachment. 2.4 Top ranking residues in 1hroA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1hroA colored by their 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. 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 in Table 1. Fig. 3. Residues in 1hroA, colored according to the cluster they belong to: Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for cluster size member the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. color residues red 16 6,7,11,15,19,22,23,33,34,36 52,53,96,97,100,101 blue 10 63,68,71,72,75,81,82,84,86 Table 1. continued 88 cluster size member continued in next column color residues Table 1. Clusters of top ranking residues in 1hroA. 2 2.4.2 Overlap with known functional surfaces at 25% coverage. Table 3. continued The name of the ligand is composed of the source PDB identifier res type disruptive and the heteroatom name used in that file. mutations HEM binding site. Table 2 lists the top 25% of residues at the 68 L (YR)(TH)(SKECG)(FQWD) interface with 1hroAHEM1 (hem). The following table (Table 3) sug- 71 Y (K)(Q)(EM)(NR) gests possible disruptive replacements for these residues (see Section 72 I (YR)(TH)(SKECG)(FQWD) 3.6). 36 L (Y)(T)(R)(H) 53 S (KR)(FQMWH)(NELPI)(Y) Table 2. 86 Y (K)(Q)(EM)(NR) res type subst’s cvg noc/ dist (%) bb (A˚ ) Table 3. List of disruptive mutations for the top 25% of residues in 22 C C(100) 0.11 29/10 1.76 1hroA, that are at the interface with HEM. 23 H H(100) 0.11 84/0 1.97 33 G G(100) 0.11 12/12 3.87 34 P P(100) 0.11 19/1 3.77 52 Y Y(100) 0.11 27/6 2.52 63 W W(100) 0.11 36/0 2.93 82 T T(100) 0.11 16/7 2.75 84 M M(100) 0.11 96/24 2.24 19 C C(96) 0.12 35/19 1.83 A(3) 68 L L(96) 0.13 3/0 3.46 I(3) 71 Y Y(67) 0.16 32/0 3.59 F(32) 72 I L(90) 0.20 5/0 3.65 I(9) 36 L L(87) 0.22 22/0 3.57 M(3) H(6) Q(3) 53 S T(29) 0.23 16/9 2.65 S(70) 86 Y F(83) 0.24 26/6 4.05 Y(16) Table 2. The top 25% of residues in 1hroA at the interface with HEM.(Field names: res: residue number in the PDB entry; type: amino acid Fig. 4. Residues in 1hroA, at the interface with HEM, colored by their relative type; substs: substitutions seen in the alignment; with the percentage of each importance. The ligand (HEM) is colored green. Atoms further than 30A˚ type in the bracket; noc/bb: number of contacts with the ligand, with the num- away from the geometric center of the ligand, as well as on the line of sight ber of contacts realized through backbone atoms given in the bracket; dist: to the ligand were removed. (See Appendix for the coloring scheme for the distance of closest apporach to the ligand. ) protein chain 1hroA.) Table 3. Figure 4 shows residues in 1hroA colored by their importance, at the res type disruptive interface with 1hroAHEM1. mutations 2.4.3 Possible novel functional surfaces at 25% coverage. One 22 C (KER)(FQMWHD)(NYLPI)(SVA) group of residues is conserved on the 1hroA surface, away from (or 23 H (E)(TQMD)(SNKVCLAPIG)(YR) susbtantially larger than) other functional sites and interfaces reco- 33 G (KER)(FQMWHD)(NYLPI)(SVA) gnizable in PDB entry 1hro. It is shown in Fig. 5. The right panel 34 P (YR)(TH)(SKECG)(FQWD) shows (in blue) the rest of the larger cluster this surface belongs to. 52 Y (K)(QM)(NEVLAPIR)(D) The residues belonging to this surface ”patch” are listed in Table 63 W (KE)(TQD)(SNCRG)(M) 4, while Table 5 suggests possible disruptive replacements for these 82 T (KR)(FQMWH)(NELPI)(D) residues (see Section 3.6). 84 M (Y)(TH)(SCRG)(FWD) 19 C (KER)(QHD)(FYMW)(N) continued in next column 3 Fig. 5. A possible active surface on the chain 1hroA. The larger cluster it belongs to is shown in blue. Table 4. res type substitutions(%) cvg 101 Y Y(96)F(3) 0.14 97 D D(90)N(9) 0.15 7 D D(77)S(9)N(12) 0.17 6 G G(80)A(9)P(6) 0.18 Q(3) 100 A A(90)S(6)E(3) 0.21 Fig. 6. Another possible active surface on the chain 1hroA. 96 A A(90)E(6)G(3) 0.25 Table 6. continued Table 4. Residues forming surface ”patch” in 1hroA. res type substitutions(%) cvg 84 M M(100) 0.11 88 G G(100) 0.11 Table 5. 19 C C(96)A(3) 0.12 res type disruptive 68 L L(96)I(3) 0.13 mutations 71 Y Y(67)F(32) 0.16 101 Y (K)(Q)(EM)(NR) 72 I L(90)I(9) 0.20 97 D (R)(FWH)(Y)(VCAG) 36 L L(87)M(3)H(6) 0.22 7 D (R)(FWH)(Y)(K) Q(3) 6 G (R)(E)(KH)(Y) 53 S T(29)S(70) 0.23 100 A (R)(Y)(K)(H) 86 Y F(83)Y(16) 0.24 96 A (R)(KY)(H)(E) Table 6.