Pages 1–6 1ako Evolutionary trace report by report maker November 5, 2010 4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 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 1ako): Title: Exonuclease iii from escherichia coli Compound: Mol id: 1; molecule: exonuclease iii; chain: a; ec: 3.1.11.2; engineered: yes Organism, scientific name: Escherichia Coli; 1ako contains a single unique chain 1akoA (268 residues long). 2 CHAIN 1AKOA 2.1 P09030 overview CONTENTS From SwissProt, id P09030, 100% identical to 1akoA: Description: Exodeoxyribonuclease III (EC 3.1.11.2) (Exonuclease 1 Introduction 1 III) (EXO III) (AP endonuclease VI). 2 Chain 1akoA 1 Organism, scientific name: Escherichia coli. 2.1 P09030 overview 1 Taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria; 2.2 Multiple sequence alignment for 1akoA 1 Enterobacteriales; Enterobacteriaceae; Escherichia. 2.3 Residue ranking in 1akoA 1 Function: Major apurinic-apyrimidinic endonuclease of E.coli. It 2.4 Top ranking residues in 1akoA and their position on removes the damaged DNA at cytosines and guanines by cleaving the structure 2 on the 3’ side of the AP site by a beta-elimination reaction. It exhi- 2.4.1 Clustering of residues at 25% coverage. 2 bits 3’-5’-exonuclease, 3’-phosphomonoesterase, 3’-repair diesterase 2.4.2 Possible novel functional surfaces at 25% and ribonuclease H activities. coverage. 2 Catalytic activity: Exonucleolytic cleavage in the 3’- to 5’- direction to yield nucleoside 5’-phosphates. 3 Notes on using trace results 4 Subunit: Monomer. 3.1 Coverage 4 Interaction: 3.2 Known substitutions 4 Similarity: Belongs to the DNA repair enzymes AP/exoA family. 3.3 Surface 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.4 Number of contacts 4 collaboration between the Swiss Institute of Bioinformatics and the 3.5 Annotation 4 EMBL outstation - the European Bioinformatics Institute. There are 3.6 Mutation suggestions 4 no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4 Appendix 5 4.1 File formats 5 2.2 Multiple sequence alignment for 1akoA 4.2 Color schemes used 5 For the chain 1akoA, the alignment 1akoA.msf (attached) with 97 4.3 Credits 5 sequences was used. The alignment was assembled through combi- 4.3.1 Alistat 5 nation of BLAST searching on the UniProt database and alignment 4.3.2 CE 5 using Muscle program. It can be found in the attachment to this 1 Lichtarge lab 2006 Fig. 1. Residues 1-134 in 1akoA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) Fig. 2. Residues 135-268 in 1akoA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) Fig. 3. Residues in 1akoA, colored by their relative importance. Clockwise: front, back, top and bottom views. report, under the name of 1akoA.msf. Its statistics, from the alistat program are the following: 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Format: MSF top 25% of all residues, this time colored according to clusters they Number of sequences: 97 belong to. The clusters in Fig.4 are composed of the residues listed Total number of residues: 24602 Smallest: 201 Largest: 268 Average length: 253.6 Alignment length: 268 Average identity: 34% Most related pair: 99% Most unrelated pair: 20% Most distant seq: 48% Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 1% eukaryotic ( 1% vertebrata), 95% prokaryotic, and 3% archaean sequences. The file containing the sequence descriptions can be found in the attachment, under the name 1akoA.descr. 2.3 Residue ranking in 1akoA The 1akoA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1akoA can be found in the file called 1akoA.ranks sorted in the attachment. 2.4 Top ranking residues in 1akoA and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 1akoA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for residues in the protein . Figure 3 shows residues in 1akoA colored the coloring scheme). Clockwise: front, back, top and bottom views. The by their importance: bright red and yellow indicate more conser- corresponding Pymol script is attached. ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1. 2 Table 1. cluster size member color residues red 63 5,6,7,8,9,10,12,14,18,33,34 36,42,43,58,62,63,64,65,66 88,90,107,109,111,112,113 122,125,128,150,151,153,154 155,156,160,161,178,183,184 197,200,210,211,212,213,214 215,220,225,226,227,228,229 230,251,256,257,258,259,260 261 blue 2 51,52 Table 1. Clusters of top ranking residues in 1akoA. 2.4.2 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1akoA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1ako. It is shown in Fig. 5. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Fig. 5. A possible active surface on the chain 1akoA. The larger cluster it belongs to is shown in blue. The residues belonging to this surface ”patch” are listed in Table 2, while Table 3 suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type substitutions(%) cvg antn 9 N N(98)D(1) 0.03 34 E E(100) 0.03 site 90 R R(100) 0.03 109 Y Y(100) 0.03 151 D D(100) 0.03 153 N N(100) 0.03 36 K K(92)R(7) 0.04 227 R R(98).(1) 0.06 229 D D(98).(1) 0.06 258 D D(98).(1) 0.06 259 H H(98).(1) 0.06 continued in next column 3 Table 2. continued Table 2. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn 111 P P(96)H(2)V(1) 0.08 161 I L(27)I(29)V(34) 0.23 212 W W(75)F(16)Y(8) 0.09 S(2)A(4)C(1) 251 R .(27)R(67)T(2) 0.09 M(1) S(3) 43 P S(5)P(79)Q(1) 0.24 261 P P(97)A(1).(1) 0.09 A(1)T(1)D(5) 112 Q S(9)N(60)K(1) 0.10 E(3)R(1)N(2) Q(16)R(2)A(10) .(1) 215 Y N(3)Y(92)W(1) 0.10 13 A S(6)T(15)A(56) 0.25 Q(1)S(2) V(4)K(4)L(10) 10 G G(60)S(36)N(3) 0.11 G(2)R(1) 197 D D(94).(2)E(2) 0.11 18 L G(25)V(19)L(42) 0.25 A(1) N(4)M(3)K(1) 113 G G(82)A(6)S(11) 0.12 A(1)I(2)D(1) 178 S G(34)M(4)L(18) 0.12 58 G Y(9)G(67)P(6) 0.25 H(9)C(7)S(26) S(7)E(2)A(2) 63 Y S(27)N(46)A(4) 0.13 D(3)N(1)K(1) Y(16)H(5) F(1) 128 F F(53)W(42)Y(4) 0.13 156 P H(30)L(3)P(61) 0.13 Table 2. Residues forming surface ”patch” in 1akoA. S(1)R(2)V(1) 184 R R(84)Q(5)L(2) 0.14 T(6)V(1)I(1) Table 3. 214 D S(44)D(50)N(3) 0.14 res type disruptive T(1)K(1) mutations 122 F Q(10)Y(17)F(48) 0.15 9 N (Y)(FWH)(TR)(VCAG) L(21)I(1)M(1) 34 E (FWH)(YVCARG)(T)(SNKLPI) 200 R R(92)M(1)K(1) 0.15 90 R (TD)(SYEVCLAPIG)(FMW)(N) L(1)Q(1)T(1) 109 Y (K)(QM)(NEVLAPIR)(D) P(1)D(1) 151 D (R)(FWH)(KYVCAG)(TQM) 12 R R(75)N(10)K(12) 0.16 153 N (Y)(FTWH)(SEVCARG)(MD) A(2) 36 K (Y)(T)(FW)(SVCAG) 14 R A(11)R(65)I(7) 0.16 227 R (TD)(SVCLAPIG)(YE)(FMW) H(1)C(3)W(1) 229 D (R)(FWH)(VCAG)(KY) V(10) 258 D (R)(FWH)(VCAG)(KY) 62 H Y(54)W(16)R(7) 0.17 259 H (E)(TQMD)(SNVCLAPIG)(K) H(15)F(6) 111 P (R)(Y)(T)(E) 228 I I(77)L(21).(1) 0.17 212 W (K)(E)(Q)(D) 210 F Y(63)W(5)F(29) 0.18 251 R (D)(LPI)(E)(FTYVMAW) L(1) 261 P (Y)(R)(H)(T) 230 L Y(31)H(37)F(7) 0.19 112 Q (Y)(FW)(TH)(CG) M(6)V(4)L(12) 215 Y (K)(M)(EQR)(VA) .(1) 10 G (R)(KE)(FWH)(M) 42 F L(15)F(69)I(8) 0.20 197 D (R)(H)(FW)(Y) T(3)M(1)V(3) 113 G (KR)(E)(QH)(FMW) 88 Q E(40)Q(43)S(2) 0.22 178 S (R)(K)(Q)(H) D(7)H(4)G(1) 63 Y (K)(Q)(M)(E) A(1).(1) 128 F (K)(E)(Q)(D) 220 F Y(2)W(29)F(46) 0.22 156 P (Y)(R)(T)(E) R(20).(1) 184 R (Y)(T)(D)(E) 256 P F(5)P(49)A(13) 0.22 214 D (R)(FW)(H)(Y) W(3)T(3)G(22) 122 F (KE)(T)(D)(R) S(2).(1) 200 R (TY)(D)(CG)(S) continued in next column continued in next column 4 Table 3.