Pages 1–11 2i52 Evolutionary trace report by report maker February 25, 2010

4.3.3 DSSP 10 4.3.4 HSSP 10 4.3.5 LaTex 10 4.3.6 Muscle 10 4.3.7 Pymol 10 4.4 Note about ET Viewer 10 4.5 Citing this work 10 4.6 About report maker 11 4.7 Attachments 11

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2i52): Title: Crystal structure of protein pto0218 from torridus, pfam duf372 Compound: Mol id: 1; molecule: hypothetical protein; chain: a, b, CONTENTS c, d, e, f; engineered: yes Organism, scientific name: ; 1 Introduction 1 2i52 contains a single unique chain 2i52F (119 residues long) and its homologues 2i52A, 2i52D, 2i52C, 2i52E, and 2i52B. 2 Chain 2i52F 1 2.1 Q6L2J9 overview 1 2.2 Multiple sequence alignment for 2i52F 1 2.3 Residue ranking in 2i52F 1 2.4 Top ranking residues in 2i52F and their position on the structure 1 2 CHAIN 2I52F 2.4.1 Clustering of residues at 25% coverage. 1 2.4.2 Overlap with known functional surfaces at 2.1 Q6L2J9 overview 25% coverage. 2 From SwissProt, id Q6L2J9, 97% identical to 2i52F: Description: Hypothetical protein. 3 Notes on using trace results 9 Organism, scientific name: Picrophilus torridus. 3.1 Coverage 9 : ; ; ; Thermoplas- 3.2 Known substitutions 9 matales; ; Picrophilus. 3.3 Surface 9 3.4 Number of contacts 9 3.5 Annotation 9 3.6 Mutation suggestions 9 2.2 Multiple sequence alignment for 2i52F 4 Appendix 9 For the chain 2i52F, the alignment 2i52F.msf (attached) with 30 4.1 File formats 9 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 10 database, and fragments shorter than 75% of the query as well as 4.3 Credits 10 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 10 to this report, under the name of 2i52F.msf. Its statistics, from the 4.3.2 CE 10 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 0-120 in 2i52F colored by their relative importance. (See Appendix, Fig.15, for the coloring scheme.)

Format: MSF Number of sequences: 30 Total number of residues: 1932 Smallest: 56 Largest: 119 Average length: 64.4 Alignment length: 119 Average identity: 54% Most related pair: 97% Most unrelated pair: 31% Most distant seq: 55% Fig. 2. Residues in 2i52F, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, 7% of residues show as conserved in this alignment. The alignment consists of 10% prokaryotic, and 40% archaean sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 2i52F.descr. 2.3 Residue ranking in 2i52F The 2i52F sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2i52F can be found in the file called 2i52F.ranks sorted in the attachment. 2.4 Top ranking residues in 2i52F 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 2i52F 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 2i52F, 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 color residues corresponding Pymol script is attached. red 30 12,14,15,17,19,20,21,22,23 24,25,26,27,28,29,30,31,32 34,36,45,46,48,49,52,56,57 2.4.2 Overlap with known functional surfaces at 25% coverage. 59,62,64 The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 2i52B.Table 2 lists the top 25% of residues at the Table 1. Clusters of top ranking residues in 2i52F. interface with 2i52B. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

2 Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 64 I V(93) 0.08 2/2 4.64 I(6) 46 E E(90) 0.13 13/0 2.90 A(10) 62 V I(43) 0.25 1/1 4.74 V(46) A(10)

Table 2. The top 25% of residues in 2i52F at the interface with 2i52B. (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. )

Table 3. res type disruptive mutations 64 I (YR)(H)(TKE)(SQCDG) 46 E (H)(FYWR)(CG)(TKVA) 62 V (YR)(KE)(H)(QD)

Table 3. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with 2i52B.

Fig. 4. Residues in 2i52F, at the interface with 2i52B, colored by their relative importance. 2i52B is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2i52F.)

Figure 4 shows residues in 2i52F colored by their importance, at the interface with 2i52B.

3 Glycerol binding site. By analogy with 2i52C – 2i52GOL902 interface. Table 4 lists the top 25% of residues at the interface with 2i52GOL902 (glycerol). The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 4. res type subst’s cvg noc/ dist (%) bb (A˚ ) 56 Q Q(100) 0.08 7/0 3.02 23 I I(96) 0.12 2/0 4.13 V(3) 27 A A(83) 0.23 1/0 3.89 T(10) S(6)

Table 4. The top 25% of residues in 2i52F at the interface with glyce- rol.(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. )

Fig. 5. Residues in 2i52F, at the interface with glycerol, colored by their rela- Table 5. tive importance. The ligand (glycerol) is colored green. Atoms further than res type disruptive 30A˚ away from the geometric center of the ligand, as well as on the line of mutations sight to the ligand were removed. (See Appendix for the coloring scheme for 56 Q (Y)(FTWH)(SVCAG)(D) the protein chain 2i52F.) 23 I (YR)(H)(TKE)(SQCDG) 27 A (KR)(E)(Y)(QH)

Table 5. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with glycerol.

Figure 5 shows residues in 2i52F colored by their importance, at the interface with 2i52GOL902. Chloride ion binding site. By analogy with 2i52B – 2i52CL813 interface. Table 6 lists the top 25% of residues at the interface with 2i52CL813 (chloride ion). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 14 I R(76) 0.20 4/2 3.46 A(10) K(3) I(3) P(6) 12 T T(73) 0.21 5/2 3.87 S(23) N(3)

Table 6. The top 25% of residues in 2i52F at the interface with chloride ion.(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. )

4 Table 7. Table 8. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 14 I (Y)(T)(R)(H) W(6) 12 T (R)(K)(FWH)(M) Table 8. The top 25% of residues in 2i52F at the interface with calcium Table 7. List of disruptive mutations for the top 25% of residues in 2i52F, ion.(Field names: res: residue number in the PDB entry; type: amino acid that are at the interface with chloride ion. 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 9. res type disruptive mutations 30 H (E)(TQMD)(SNKVCLAPIG)(YR) 31 Q (Y)(FTWH)(SVCAG)(D) 34 G (KER)(FQMWHD)(NYLPI)(SVA) 29 F (K)(E)(Q)(D) 32 Y (K)(Q)(E)(M)

Table 9. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with calcium ion.

Fig. 6. Residues in 2i52F, at the interface with chloride ion, colored by their relative importance. The ligand (chloride ion) 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 2i52F.)

Figure 6 shows residues in 2i52F colored by their importance, at the interface with 2i52CL813. Calcium ion binding site. Table 8 lists the top 25% of residues at the interface with 2i52CA808 (calcium ion). The following table (Table 9) suggests possible disruptive replacements for these residues (see Section 3.6). Table 8. res type subst’s cvg noc/ dist (%) bb (A˚ ) 30 H H(100) 0.08 3/3 2.87 Fig. 7. Residues in 2i52F, at the interface with calcium ion, colored by their 31 Q Q(100) 0.08 3/3 4.49 relative importance. The ligand (calcium ion) is colored green. Atoms further A˚ 34 G G(100) 0.08 2/2 4.41 than 30 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 29 F F(36) 0.13 1/1 4.59 for the protein chain 2i52F.) Y(63) 32 Y F(86) 0.14 2/2 4.86 Y(6) Figure 7 shows residues in 2i52F colored by their importance, at the continued in next column interface with 2i52CA808. Glycerol binding site. By analogy with 2i52C – 2i52GOL901 interface. Table 10 lists the top 25% of residues at the interface

5 with 2i52GOL901 (glycerol). The following table (Table 11) sug- Figure 8 shows residues in 2i52F colored by their importance, at the gests possible disruptive replacements for these residues (see Section interface with 2i52GOL901. 3.6). Interface with 2i52E1.Table 12 lists the top 25% of residues at the interface with 2i52E1. The following table (Table 13) suggests Table 10. possible disruptive replacements for these residues (see Section 3.6). res type subst’s cvg noc/ dist Table 12. A˚ (%) bb ( ) res type subst’s cvg noc/ dist 30 H H(100) 0.08 15/0 2.81 (%) bb (A˚ ) 29 F F(36) 0.13 16/0 3.81 19 F F(100) 0.08 94/24 3.32 Y(63) 20 E E(100) 0.08 1/0 4.97 26 G G(100) 0.08 29/29 3.76 Table 10. The top 25% of residues in 2i52F at the interface with glyce- 30 H H(100) 0.08 60/10 2.87 rol.(Field names: res: residue number in the PDB entry; type: amino acid type; 31 Q Q(100) 0.08 29/8 2.73 substs: substitutions seen in the alignment; with the percentage of each type 57 P P(100) 0.08 3/0 4.07 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 22 G G(93) 0.11 46/46 3.21 of closest apporach to the ligand. ) A(6) 23 I I(96) 0.12 42/21 3.23 V(3) 21 A G(36) 0.16 8/6 3.99 Table 11. A(59) res type disruptive S(3) mutations 25 L L(66) 0.18 5/0 3.67 30 H (E)(TQMD)(SNKVCLAPIG)(YR) F(16) 29 F (K)(E)(Q)(D) M(16) 14 I R(76) 0.20 9/0 3.68 A(10) Table 11. List of disruptive mutations for the top 25% of residues in K(3) 2i52F, that are at the interface with glycerol. I(3) P(6) 15 Q E(63) 0.23 46/14 3.62 D(20) Q(16) 27 A A(83) 0.23 34/26 3.36 T(10) S(6)

Table 12. The top 25% of residues in 2i52F at the interface with 2i52E1. (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. )

Table 13. res type disruptive mutations 19 F (KE)(TQD)(SNCRG)(M) 20 E (FWH)(YVCARG)(T)(SNKLPI) 26 G (KER)(FQMWHD)(NYLPI)(SVA) 30 H (E)(TQMD)(SNKVCLAPIG)(YR) 31 Q (Y)(FTWH)(SVCAG)(D) 57 P (YR)(TH)(SKECG)(FQWD) 22 G (KER)(QHD)(FYMW)(N) Fig. 8. Residues in 2i52F, at the interface with glycerol, colored by their rela- 23 I (YR)(H)(TKE)(SQCDG) tive importance. The ligand (glycerol) is colored green. Atoms further than continued in next column 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 2i52F.)

6 Table 13. continued res type disruptive mutations 21 A (KR)(E)(Y)(QH) 25 L (YR)(T)(H)(KECG) 14 I (Y)(T)(R)(H) 15 Q (Y)(FWH)(T)(VCAG) 27 A (KR)(E)(Y)(QH)

Table 13. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with 2i52E1.

Fig. 9. Residues in 2i52F, at the interface with 2i52E1, colored by their rela- tive importance. 2i52E1 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2i52F.)

Figure 9 shows residues in 2i52F colored by their importance, at the interface with 2i52E1. Calcium ion binding site. Table 14 lists the top 25% of resi- dues at the interface with 2i52CA809 (calcium ion). The following table (Table 15) suggests possible disruptive replacements for these residues (see Section 3.6). Table 14. res type subst’s cvg noc/ dist (%) bb (A˚ ) 31 Q Q(100) 0.08 1/0 4.89

Table 14. The top 25% of residues in 2i52F at the interface with calcium ion.(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. )

7 Table 15. type in the bracket; noc/bb: number of contacts with the ligand, with the num- res type disruptive ber of contacts realized through backbone atoms given in the bracket; dist: mutations distance of closest apporach to the ligand. ) 31 Q (Y)(FTWH)(SVCAG)(D)

Table 15. List of disruptive mutations for the top 25% of residues in Table 17. 2i52F, that are at the interface with calcium ion. res type disruptive mutations 56 Q (Y)(FTWH)(SVCAG)(D) 59 V (YR)(KE)(H)(D)

Table 17. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with calcium ion.

Fig. 10. Residues in 2i52F, at the interface with calcium ion, colored by their relative importance. The ligand (calcium ion) 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 2i52F.) Fig. 11. Residues in 2i52F, at the interface with calcium ion, colored by their relative importance. The ligand (calcium ion) is colored green. Atoms further A˚ Figure 10 shows residues in 2i52F colored by their importance, at the than 30 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 interface with 2i52CA809. for the protein chain 2i52F.) Calcium ion binding site. By analogy with 2i52B – 2i52CA810 interface. Table 16 lists the top 25% of residues at the interface with 2i52CA810 (calcium ion). The following table (Table 17) suggests Figure 11 shows residues in 2i52F colored by their importance, at the possible disruptive replacements for these residues (see Section 3.6). interface with 2i52CA810. Calcium ion binding site. Table 18 lists the top 25% of resi- Table 16. dues at the interface with 2i52CA801 (calcium ion). The following res type subst’s cvg noc/ dist table (Table 19) suggests possible disruptive replacements for these (%) bb (A˚ ) residues (see Section 3.6). 56 Q Q(100) 0.08 2/2 4.66 Table 18. 59 V V(50) 0.19 1/1 4.84 res type subst’s cvg noc/ dist I(29) (%) bb (A˚ ) C(16) 64 I V(93) 0.08 1/1 4.52 M(3) I(6) 46 E E(90) 0.13 5/0 2.47 Table 16. The top 25% of residues in 2i52F at the interface with calcium continued in next column ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each

8 Table 18. continued Table 20. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) A(10) 31 Q Q(100) 0.08 50/4 2.92 34 G G(100) 0.08 14/14 3.31 Table 18. The top 25% of residues in 2i52F at the interface with calcium 36 P P(100) 0.08 37/10 3.52 ion.(Field names: res: residue number in the PDB entry; type: amino acid 56 Q Q(100) 0.08 30/4 3.47 type; substs: substitutions seen in the alignment; with the percentage of each 57 P P(100) 0.08 10/1 3.84 type in the bracket; noc/bb: number of contacts with the ligand, with the num- 32 Y F(86) 0.14 17/5 3.28 ber of contacts realized through backbone atoms given in the bracket; dist: Y(6) distance of closest apporach to the ligand. ) W(6) 48 F A(86) 0.15 78/13 3.18 S(6) Table 19. C(3) res type disruptive F(3) mutations 45 A L(80) 0.18 15/11 3.30 64 I (YR)(H)(TKE)(SQCDG) V(13) 46 E (H)(FYWR)(CG)(TKVA) A(3) M(3) Table 19. List of disruptive mutations for the top 25% of residues in 52 S A(36) 0.22 32/20 3.34 2i52F, that are at the interface with calcium ion. S(63)

Table 20. The top 25% of residues in 2i52F at the interface with 2i52E. (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. )

Table 21. res type disruptive mutations 31 Q (Y)(FTWH)(SVCAG)(D) 34 G (KER)(FQMWHD)(NYLPI)(SVA) 36 P (YR)(TH)(SKECG)(FQWD) 56 Q (Y)(FTWH)(SVCAG)(D) 57 P (YR)(TH)(SKECG)(FQWD) 32 Y (K)(Q)(E)(M) 48 F (K)(E)(Q)(DR) 45 A (Y)(R)(KE)(H) 52 S (KR)(QH)(FYEMW)(N)

Table 21. List of disruptive mutations for the top 25% of residues in 2i52F, that are at the interface with 2i52E.

Fig. 12. Residues in 2i52F, at the interface with calcium ion, colored by their Figure 13 shows residues in 2i52F colored by their importance, at the relative importance. The ligand (calcium ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line interface with 2i52E. of sight to the ligand were removed. (See Appendix for the coloring scheme Interface with 2i52F1.Table 22 lists the top 25% of residues at for the protein chain 2i52F.) the interface with 2i52F1. The following table (Table 23) suggests possible disruptive replacements for these residues (see Section 3.6).

Figure 12 shows residues in 2i52F colored by their importance, at the Table 22. interface with 2i52CA801. res type subst’s cvg noc/ dist Interface with 2i52E.Table 20 lists the top 25% of residues at (%) bb (A˚ ) the interface with 2i52E. The following table (Table 21) suggests 30 H H(100) 0.08 8/0 3.36 possible disruptive replacements for these residues (see Section 3.6). 34 G G(100) 0.08 7/7 3.36 continued in next column

9 Fig. 13. Residues in 2i52F, at the interface with 2i52E, colored by their rela- Fig. 14. Residues in 2i52F, at the interface with 2i52F1, colored by their rela- tive importance. 2i52E is shown in backbone representation (See Appendix tive importance. 2i52F1 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2i52F.) for the coloring scheme for the protein chain 2i52F.)

3 NOTES ON USING TRACE RESULTS Table 22. continued res type subst’s cvg noc/ dist 3.1 Coverage (%) bb (A˚ ) Trace results are commonly expressed in terms of coverage: the resi- 36 P P(100) 0.08 1/0 4.90 due is important if its “coverage” is small - that is if it belongs to 29 F F(36) 0.13 6/0 3.62 some small top percentage of residues [100% is all of the residues Y(63) in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or Table 22. The top 25% of residues in 2i52F at the interface with 2i52F1. to some nearby percentage), sorted by the strength of the presumed (Field names: res: residue number in the PDB entry; type: amino acid type; evolutionary pressure. (I.e., the smaller the coverage, the stronger the substs: substitutions seen in the alignment; with the percentage of each type pressure on the residue.) Starting from the top of that list, mutating a in the bracket; noc/bb: number of contacts with the ligand, with the number of couple of residues should affect the protein somehow, with the exact contacts realized through backbone atoms given in the bracket; dist: distance effects to be determined experimentally. of closest apporach to the ligand. ) 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types may be interchangeable at that position in the protein, so if one wants Table 23. to affect the protein by a point mutation, they should be avoided. For res type disruptive example if the substitutions are “RVK” and the original protein has mutations an R at that position, it is advisable to try anything, but RVK. Conver- 30 H (E)(TQMD)(SNKVCLAPIG)(YR) sely, when looking for substitutions which will not affect the protein, 34 G (KER)(FQMWHD)(NYLPI)(SVA) one may try replacing, R with K, or (perhaps more surprisingly), with 36 P (YR)(TH)(SKECG)(FQWD) V. The percentage of times the substitution appears in the alignment 29 F (K)(E)(Q)(D) 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 23. List of disruptive mutations for the top 25% of residues in guide - due to rounding errors these percentages often do not add up 2i52F, that are at the interface with 2i52F1. to 100%.

3.3 Surface Figure 14 shows residues in 2i52F colored by their importance, at the To detect candidates for novel functional interfaces, first we look for interface with 2i52F1. residues that are solvent accessible (according to DSSP program) by

10 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, COVERAGE the neighboring residues which are not surface accessible might be

equally important in maintaining the interaction specificity - they V should not be automatically dropped from consideration when choo- 100% 50% 30% 5% sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across V the interface, as well as how many of them are realized through the RELATIVE IMPORTANCE backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . Fig. 15. Coloring scheme used to color residues by their relative importance. 3.5 Annotation If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller • gaps Mutation suggestions are completely heuristic and based on comple- percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 15. disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will 4.3 Credits choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4 APPENDIX of residues, the average and range of the sequence lengths, and the 4.1 File formats alignment length (e.g. including gap characters). Also shown are Files with extension “ranks sorted” are the actual trace results. The some percent identities. A percent pairwise alignment identity is defi- fields in the table in this file: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two • alignment# number of the position in the alignment sequences. The ”average percent identity”, ”most related pair”, and • residue# residue number in the PDB file ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • type amino acid type seq” is calculated by finding the maximum pairwise identity (best • rank rank of the position according to older version of ET relative) for all N sequences, then finding the minimum of these N • variability has two subfields: numbers (hence, the most outlying sequence). alistat is copyrighted 1. number of different amino acids appearing in in this column by HHMI/Washington University School of Medicine, 1992-2001, of the alignment and freely distributed under the GNU General Public License.

11 4.3.2 CE To map ligand binding sites from different Evolution-Entropy Hybrid Methods for Ranking of Protein Residues source structures, report maker uses the CE program: by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- ”Protein structure alignment by incremental combinatorial extension tionary Trace Method Defines Binding Surfaces Common to Protein (CE) of the optimal path . Protein Engineering 11(9) 739-747. Families” J. Mol. Bio. 257: 342-358. report maker itself is described in Mihalek I., I. Res and O. 4.3.3 DSSP In this work a residue is considered solvent accessi- Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type ble if the DSSP program finds it exposed to water by at least 10A˚ 2, of service for comparative analysis of proteins.” Bioinformatics which is roughly the area needed for one water molecule to come in 22:1656-7. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, 4.6 About report maker http://www.cmbi.kun.nl/gv/dssp/descrip.html. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.4 HSSP Whenever available, report maker uses HSSP ali- is copyrighted by Lichtarge Lab, Baylor College of Medicine, gnment as a starting point for the analysis (sequences shorter than Houston. 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- 4.7 Attachments sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. The following files should accompany this report: http://swift.cmbi.kun.nl/swift/hssp/ • 4.3.5 LaTex The text for this report was processed using LATEX; 2i52F.complex.pdb - coordinates of 2i52F with all of its interac- Leslie Lamport, “LaTeX: A Document Preparation System Addison- ting partners Wesley,” Reading, Mass. (1986). • 2i52F.etvx - ET viewer input file for 2i52F 4.3.6 Muscle When making alignments “from scratch”, report • 2i52F.cluster report.summary - Cluster report summary for maker uses Muscle alignment program: Edgar, Robert C. (2004), 2i52F ”MUSCLE: multiple sequence alignment with high accuracy and • 2i52F.ranks - Ranks file in sequence order for 2i52F high throughput.” Nucleic Acids Research 32(5), 1792-97. • 2i52F.clusters - Cluster descriptions for 2i52F http://www.drive5.com/muscle/ • 2i52F.msf - the multiple sequence alignment used for the chain 2i52F 4.3.7 Pymol The figures in this report were produced using Pymol. The scripts can be found in the attachment. Pymol • 2i52F.descr - description of sequences used in 2i52F msf is an open-source application copyrighted by DeLano Scien- • 2i52F.ranks sorted - full listing of residues and their ranking for tific LLC (2005). For more information about Pymol see 2i52F http://pymol.sourceforge.net/. (Note for Windows • 2i52F.2i52B.if.pml - Pymol script for Figure 4 users: the attached package needs to be unzipped for Pymol to read • 2i52F.cbcvg - used by other 2i52F – related pymol scripts the scripts and launch the viewer.) • 2i52F.2i52GOL902.if.pml - Pymol script for Figure 5 4.4 Note about ET Viewer • 2i52F.2i52CL813.if.pml - Pymol script for Figure 6 Dan Morgan from the Lichtarge lab has developed a visualization • 2i52F.2i52CA808.if.pml - Pymol script for Figure 7 tool specifically for viewing trace results. If you are interested, please visit: • 2i52F.2i52GOL901.if.pml - Pymol script for Figure 8 • 2i52F.2i52E1.if.pml - Pymol script for Figure 9 http://mammoth.bcm.tmc.edu/traceview/ • 2i52F.2i52CA809.if.pml - Pymol script for Figure 10 The viewer is self-unpacking and self-installing. Input files to be used • 2i52F.2i52CA810.if.pml - Pymol script for Figure 11 with ETV (extension .etvx) can be found in the attachment to the • main report. 2i52F.2i52CA801.if.pml - Pymol script for Figure 12 • 2i52F.2i52E.if.pml - Pymol script for Figure 13 4.5 Citing this work • 2i52F.2i52F1.if.pml - Pymol script for Figure 14 The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of

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