Pages 1–9 1aq6 Evolutionary trace report by report maker June 5, 2010

4.3.1 Alistat 8 4.3.2 CE 8 4.3.3 DSSP 8 4.3.4 HSSP 8 4.3.5 LaTex 8 4.3.6 Muscle 8 4.3.7 Pymol 8 4.4 Note about ET Viewer 8 4.5 Citing this work 8 4.6 About report maker 9 4.7 Attachments 9

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1aq6): Title: Structure of l-2-haloacid dehalogenase from xanthobacter autotrophicus Compound: Mol id: 1; molecule: l-2-haloacid dehalogenase; chain: a, b; ec: 3.8.1.2; other details: substrate analogue formate present in both active sites Organism, scientific name: Xanthobacter Autotrophicus; 1aq6 contains a single unique chain 1aq6A (245 residues long) and CONTENTS its homologue 1aq6B. 1 Introduction 1 2 CHAIN 1AQ6A 2 Chain 1aq6A 1 2.1 Q60099 overview 2.1 Q60099 overview 1 2.2 Multiple sequence alignment for 1aq6A 1 From SwissProt, id Q60099, 100% identical to 1aq6A: 2.3 Residue ranking in 1aq6A 1 Description: (S)-2-haloacid dehalogenase (EC 3.8.1.2) (2- 2.4 Top ranking residues in 1aq6A and their position on haloalkanoic acid dehalogenase) (L-2-haloacid dehalogenase) the structure 1 (Halocarboxylic acid halidohydrolase). 2.4.1 Clustering of residues at 25% coverage. 2 Organism, scientific name: Xanthobacter autotrophicus. 2.4.2 Overlap with known functional surfaces at Taxonomy: Bacteria; Proteobacteria; Alphaproteobacteria; Rhizo- 25% coverage. 3 biales; Xanthobacteraceae; Xanthobacter. 2.4.3 Possible novel functional surfaces at 25% Function: Catalyzes the hydrolytic dehalogenation of small L-2- coverage. 5 haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. Active with 2-halogenated carboxylic acids and converts only 3 Notes on using trace results 7 the L-isomer of 2-chloropropionic acid with inversion of configura- 3.1 Coverage 7 tion to produce D-lactate. 3.2 Known substitutions 7 Catalytic activity: (S)-2-haloacid + H(2)O = (R)-2-hydroxyacid + 3.3 Surface 7 halide. 3.4 Number of contacts 7 Biophysicochemical properties: 3.5 Annotation 7 pH dependence: Optimum pH is 9.5; 3.6 Mutation suggestions 7 Subunit: Homodimer. Similarity: Belongs to the (S)-2-haloalkanoic acid dehalogenase 4 Appendix 7 family. 4.1 File formats 7 About: This Swiss-Prot entry is copyright. It is produced through a 4.2 Color schemes used 8 collaboration between the Swiss Institute of Bioinformatics and the 4.3 Credits 8 EMBL outstation - the European Bioinformatics Institute. There are

1 Lichtarge lab 2006 in 1aq6A can be found in the file called 1aq6A.ranks sorted in the attachment. 2.4 Top ranking residues in 1aq6A and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1aq6A colored by their importance: bright red and yellow indicate more conser- Fig. 1. Residues 1-122 in 1aq6A colored by their relative importance. (See ved/important residues (see Appendix for the coloring scheme). A Appendix, Fig.10, for the coloring scheme.) Pymol script for producing this figure can be found in the attachment.

Fig. 2. Residues 123-245 in 1aq6A colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.) 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 1aq6A For the chain 1aq6A, the alignment 1aq6A.msf (attached) with 36 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 1aq6A.msf. Its statistics, from the alistat program are the following:

Format: MSF Fig. 3. Residues in 1aq6A, colored by their relative importance. Clockwise: Number of sequences: 36 front, back, top and bottom views. Total number of residues: 7812 Smallest: 190 Largest: 245 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Average length: 217.0 top 25% of all residues, this time colored according to clusters they Alignment length: 245 belong to. The clusters in Fig.4 are composed of the residues listed Average identity: 32% in Table 1. Most related pair: 96% Most unrelated pair: 15% Table 1. Most distant seq: 30% cluster size member color residues red 60 7,8,10,11,12,13,15,38,39,42 Furthermore, <1% of residues show as conserved in this ali- 43,44,45,47,48,51,55,58,62 gnment. 65,66,89,92,95,97,102,105 The alignment consists of 5% eukaryotic ( 5% fungi), 80% 113,114,115,116,117,121,125 prokaryotic, and 11% archaean sequences. (Descriptions of some 129,130,134,135,139,140,141 sequences were not readily available.) The file containing the 146,147,148,150,153,169,170 sequence descriptions can be found in the attachment, under the name 171,172,173,175,176,179,180 1aq6A.descr. 184,188,189,190,192 2.3 Residue ranking in 1aq6A Table 1. Clusters of top ranking residues in 1aq6A. The 1aq6A sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) M(5) G(2) 44 E E(75) 0.18 31/0 2.90 Q(5) A(2) P(2) G(5) D(5) H(2) 150 P D(5) 0.19 12/2 3.57 P(69) R(2) N(8) L(5) S(5) V(2) 175 F W(61) 0.20 7/1 3.75 F(19) Y(2) Fig. 4. Residues in 1aq6A, colored according to the cluster they belong to: R(2) red, followed by blue and yellow are the largest clusters (see Appendix for K(5) the coloring scheme). Clockwise: front, back, top and bottom views. The H(2) corresponding Pymol script is attached. G(2) V(2) 146 F Y(50) 0.21 3/3 4.29 2.4.2 Overlap with known functional surfaces at 25% coverage. F(36) The name of the ligand is composed of the source PDB identifier K(2) and the heteroatom name used in that file. G(5) Interface with 1aq6B.Table 2 lists the top 25% of residues at the S(2) interface with 1aq6B. The following table (Table 3) suggests possible I(2) disruptive replacements for these residues (see Section 3.6). 51 L L(69) 0.22 71/23 3.49 Table 2. S(5) res type subst’s cvg noc/ dist I(8) (%) bb (A˚ ) V(2) 147 K K(100) 0.00 2/2 4.19 R(2) 148 P P(83) 0.05 35/16 3.68 Q(2) T(16) M(5) 179 G G(88) 0.05 5/5 3.72 W(2) A(11) 48 L L(58) 0.23 56/6 3.33 45 Y Y(86) 0.12 75/2 2.71 R(11) R(2) T(5) H(2) Y(2) S(2) I(5) N(2) V(13) E(2) Q(2) 47 W W(63) 0.16 50/6 3.62 L(5) Table 2. The top 25% of residues in 1aq6A at the interface with 1aq6B. E(2) (Field names: res: residue number in the PDB entry; type: amino acid type; Q(5) substs: substitutions seen in the alignment; with the percentage of each type A(8) 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 F(2) of closest apporach to the ligand. ) R(2) continued in next column

3 Table 3. Table 4. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 147 K (Y)(FTW)(SVCAG)(HD) V(2) 148 P (R)(YH)(K)(E) W(5) 179 G (KER)(QHD)(FYMW)(N) F(2) 45 Y (K)(M)(Q)(VA) Q(2) 47 W (E)(K)(T)(D) 44 E (H)(FW)(YR)(CG) Table 4. The top 25% of residues in 1aq6A at the interface with formic 150 P (Y)(R)(H)(T) acid.(Field names: res: residue number in the PDB entry; type: amino acid 175 F (E)(K)(D)(TQ) type; substs: substitutions seen in the alignment; with the percentage of each 146 F (KE)(Q)(D)(R) type in the bracket; noc/bb: number of contacts with the ligand, with the num- 51 L (Y)(R)(T)(H) ber of contacts realized through backbone atoms given in the bracket; dist: 48 L (Y)(R)(H)(T) distance of closest apporach to the ligand. )

Table 3. List of disruptive mutations for the top 25% of residues in 1aq6A, that are at the interface with 1aq6B. Table 5. res type disruptive mutations 39 R (TD)(E)(Y)(SCG)

Table 5. List of disruptive mutations for the top 25% of residues in 1aq6A, that are at the interface with formic acid.

Fig. 5. Residues in 1aq6A, at the interface with 1aq6B, colored by their rela- tive importance. 1aq6B is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1aq6A.)

Figure 5 shows residues in 1aq6A colored by their importance, at the interface with 1aq6B. Formic acid binding site. Table 4 lists the top 25% of residues Fig. 6. Residues in 1aq6A, at the interface with formic acid, colored by their at the interface with 1aq6FMT3 (formic acid). The following table relative importance. The ligand (formic acid) is colored green. Atoms further (Table 5) suggests possible disruptive replacements for these residues than 30A˚ away from the geometric center of the ligand, as well as on the line (see Section 3.6). of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1aq6A.) Table 4. res type subst’s cvg noc/ dist (%) bb (A˚ ) Figure 6 shows residues in 1aq6A colored by their importance, at the 39 R R(86) 0.11 14/4 3.34 interface with 1aq6FMT3. continued in next column Formic acid binding site. Table 6 lists the top 25% of residues at the interface with 1aq6FMT2 (formic acid). The following table

4 (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 147 K K(100) 0.00 7/0 3.36 115 N N(94) 0.01 15/7 2.90 D(5) 8 D D(97) 0.04 13/4 2.84 .(2) 114 S S(86) 0.04 15/9 2.56 T(13) 116 G G(88) 0.07 2/2 4.71 A(2) S(5) I(2) 113 L L(72) 0.15 4/4 4.45 F(8) V(8) A(2) I(8) 10 Y Y(75) 0.16 18/8 3.15 Fig. 7. Residues in 1aq6A, at the interface with formic acid, colored by their D(5) relative importance. The ligand (formic acid) is colored green. Atoms further A L(2) than 30 ˚ away from the geometric center of the ligand, as well as on the line .(2) of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1aq6A.) F(5) V(2) N(2) susbtantially larger than) other functional sites and interfaces reco- S(2) gnizable in PDB entry 1aq6. It is shown in Fig. 8. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Table 6. The top 25% of residues in 1aq6A at the interface with formic acid.(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 7. res type disruptive mutations 147 K (Y)(FTW)(SVCAG)(HD) 115 N (Y)(FWH)(TR)(VCAG) 8 D (R)(FWH)(VCAG)(KY) Fig. 8. A possible active surface on the chain 1aq6A. The larger cluster it 114 S (KR)(FQMWH)(NELPI)(Y) belongs to is shown in blue. 116 G (R)(K)(E)(H) 113 L (R)(Y)(TKEH)(SQCDG) 10 Y (K)(QR)(M)(E) The residues belonging to this surface ”patch” are listed in Table 8, while Table 9 suggests possible disruptive replacements for these residues (see Section 3.6). Table 7. List of disruptive mutations for the top 25% of residues in 1aq6A, that are at the interface with formic acid. Table 8. res type substitutions(%) cvg 147 K K(100) 0.00 Figure 7 shows residues in 1aq6A colored by their importance, at the 115 N N(94)D(5) 0.01 interface with 1aq6FMT2. 92 L L(91)Y(5)I(2) 0.02 2.4.3 Possible novel functional surfaces at 25% coverage. One continued in next column group of residues is conserved on the 1aq6A surface, away from (or

5 Table 8. continued Table 8. continued res type substitutions(%) cvg res type substitutions(%) cvg 8 D D(97).(2) 0.04 148 P P(83)T(16) 0.05 Table 8. Residues forming surface ”patch” in 1aq6A. 179 G G(88)A(11) 0.05 192 R R(88)Y(2)C(2) 0.07 P(5) 139 S S(83)T(5)I(8) 0.08 Table 9. L(2) res type disruptive 11 G G(91)D(2)E(5) 0.09 mutations 173 N N(80)R(2)H(11) 0.09 147 K (Y)(FTW)(SVCAG)(HD) S(5) 115 N (Y)(FWH)(TR)(VCAG) 172 S S(66)G(5)D(13) 0.10 92 L (R)(Y)(T)(KH) A(8)R(2)N(2) 8 D (R)(FWH)(VCAG)(KY) 15 D D(83)S(5)L(2) 0.11 148 P (R)(YH)(K)(E) R(5)T(2) 179 G (KER)(QHD)(FYMW)(N) 39 R R(86)V(2)W(5) 0.11 192 R (D)(E)(T)(SYVLAPI) F(2)Q(2) 139 S (R)(K)(H)(FQW) 45 Y Y(86)R(2)H(2) 0.12 11 G (R)(FKWH)(Y)(Q) S(2)N(2)E(2) 173 N (Y)(T)(FEW)(VCAHG) 97 D E(44)D(52)N(2) 0.13 172 S (R)(K)(FWH)(M) 55 Y Y(80)H(5)S(2) 0.14 15 D (R)(FWH)(K)(Y) .(2)E(5)D(2) 39 R (TD)(E)(Y)(SCG) 10 Y Y(75)D(5)L(2) 0.16 45 Y (K)(M)(Q)(VA) .(2)F(5)V(2) 97 D (R)(FWH)(Y)(VCAG) N(2)S(2) 55 Y (K)(Q)(M)(R) 47 W W(63)L(5)E(2) 0.16 10 Y (K)(QR)(M)(E) Q(5)A(8)F(2) 47 W (E)(K)(T)(D) R(2)M(5)G(2) 44 E (H)(FW)(YR)(CG) 44 E E(75)Q(5)A(2) 0.18 141 D (R)(FWH)(KY)(VA) P(2)G(5)D(5) 150 P (Y)(R)(H)(T) H(2) 175 F (E)(K)(D)(TQ) 141 D D(55)E(30)G(5) 0.19 117 A (R)(K)(Y)(H) K(2)A(2)Y(2) 51 L (Y)(R)(T)(H) 150 P D(5)P(69)R(2) 0.19 189 R (D)(E)(T)(S) N(8)L(5)S(5) 48 L (Y)(R)(H)(T) V(2) 190 V (R)(KE)(Y)(QHD) 175 F W(61)F(19)Y(2) 0.20 95 Y (K)(Q)(E)(M) R(2)K(5)H(2) G(2)V(2) Table 9. Disruptive mutations for the surface patch in 1aq6A. 117 A S(52)T(11)N(13) 0.21 D(8)V(5)A(8) Another group of surface residues is shown in Fig.9. The right panel 51 L L(69)S(5)I(8) 0.22 shows (in blue) the rest of the larger cluster this surface belongs to. V(2)R(2)Q(2) M(5)W(2) 189 R W(66)R(8)Y(5) 0.22 C(2)F(11)L(5) 48 L L(58)R(11)T(5) 0.23 Y(2)I(5)V(13) Q(2) 190 V I(41)V(44)F(2) 0.23 A(2)L(5)C(2) 95 Y F(30)Y(52)V(2) 0.24 W(5)H(8) continued in next column

Fig. 9. Another possible active surface on the chain 1aq6A. The larger cluster it belongs to is shown in blue.

6 The residues belonging to this surface ”patch” are listed in Table 10, is given in the immediately following bracket. No percentage is given while Table 11 suggests possible disruptive replacements for these in the cases when it is smaller than 1%. This is meant to be a rough residues (see Section 3.6). guide - due to rounding errors these percentages often do not add up to 100%. Table 10. res type substitutions(%) cvg 3.3 Surface 135 D D(69)K(11)H(2) 0.15 To detect candidates for novel functional interfaces, first we look for S(5)E(8)N(2) residues that are solvent accessible (according to DSSP program) by 2 134 F F(61)L(27)W(5) 0.18 at least 10A˚ , which is roughly the area needed for one water mole- V(2)I(2) cule to come in the contact with the residue. Furthermore, we require 125 V V(52)I(5)L(25) 0.22 that these residues form a “cluster” of residues which have neighbor A(8)T(2)K(2) within 5A˚ from any of their heavy atoms. .(2) Note, however, that, if our picture of protein evolution is correct, 129 G G(61)P(5)K(5) 0.24 the neighboring residues which are not surface accessible might be E(11)D(2)N(2) equally important in maintaining the interaction specificity - they A(11) should not be automatically dropped from consideration when choo- 130 L I(11)M(16)D(2) 0.24 sing the set for mutagenesis. (Especially if they form a cluster with L(61)V(2)W(2) the surface residues.) H(2) 3.4 Number of contacts Table 10. Residues forming surface ”patch” in 1aq6A. Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across the interface, as well as how many of them are realized through the Table 11. backbone atoms (if all or most contacts are through the backbone, res type disruptive mutation presumably won’t have strong impact). Two heavy atoms mutations are considered to be “in contact” if their centers are closer than 5A˚ . 135 D (R)(FW)(H)(Y) 134 F (KE)(T)(R)(QD) 3.5 Annotation 125 V (Y)(R)(E)(K) If the residue annotation is available (either from the pdb file or 129 G (R)(H)(FW)(Y) from other sources), another column, with the header “annotation” 130 L (R)(Y)(T)(H) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 11. Disruptive mutations for the surface patch in 1aq6A. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- 3.1 Coverage mentarity with the substitutions found in the alignment. Note that Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino couple of residues should affect the protein somehow, with the exact acid, and they are grouped in round brackets if they appear equally effects to be determined experimentally. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 4 APPENDIX an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, 4.1 File formats one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file:

7 ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N COVERAGE numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001,

V and freely distributed under the GNU General Public License. 100% 50% 30% 5% 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747.

V 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 RELATIVE IMPORTANCE 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 the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Fig. 10. Coloring scheme used to color residues by their relative importance. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 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 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de • rank rank of the position according to older version of ET Daruvar, and C. Sander. ”The HSSP database of protein structure- • variability has two subfields: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1. number of different amino acids appearing in in this column of the alignment http://swift.cmbi.kun.nl/swift/hssp/ 2. their type 4.3.5 LaTex The text for this report was processed using LATEX; • rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 10. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of

8 Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1aq6A.etvx - ET viewer input file for 1aq6A by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1aq6A.cluster report.summary - Cluster report summary for of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 1aq6A tionary Trace Method Defines Binding Surfaces Common to Protein • 1aq6A.ranks - Ranks file in sequence order for 1aq6A Families” J. Mol. Bio. 257: 342-358. report maker itself is described in Mihalek I., I. Res and O. • 1aq6A.clusters - Cluster descriptions for 1aq6A Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1aq6A.msf - the multiple sequence alignment used for the chain of service for comparative analysis of proteins.” Bioinformatics 1aq6A 22:1656-7. • 1aq6A.descr - description of sequences used in 1aq6A msf 4.6 About report maker • 1aq6A.ranks sorted - full listing of residues and their ranking report maker was written in 2006 by Ivana Mihalek. The 1D ran- for 1aq6A king visualization program was written by Ivica Res.ˇ report maker • 1aq6A.1aq6B.if.pml - Pymol script for Figure 5 is copyrighted by Lichtarge Lab, Baylor College of Medicine, • 1aq6A.cbcvg - used by other 1aq6A – related pymol scripts Houston. • 1aq6A.1aq6FMT3.if.pml - Pymol script for Figure 6 4.7 Attachments • 1aq6A.1aq6FMT2.if.pml - Pymol script for Figure 7 The following files should accompany this report: • 1aq6A.complex.pdb - coordinates of 1aq6A with all of its interacting partners

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