Pages 1–9 1j71 Evolutionary trace report by report maker September 24, 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 8 4.7 Attachments 9

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1j71): Title: Structure of the extracellular aspartic proteinase from candida tropicalis yeast. Compound: Mol id: 1; molecule: aspartic proteinase; chain: a; synonym: candidapepsin; ec: 3.4.23.24; mol id: 2; molecule: tetra- peptide thr-ile-thr-ser; chain: b Organism, scientific name: Unidentified CONTENTS 1j71 contains a single unique chain 1j71A (334 residues long). 1 Introduction 1 Chain 1j71B is too short (4 residues) to permit statistically significant analysis, and was treated as a peptide ligand. 2 Chain 1j71A 1 2.1 Q00663 overview 1 2.2 Multiple sequence alignment for 1j71A 1 2 CHAIN 1J71A 2.3 Residue ranking in 1j71A 1 2.1 Q00663 overview 2.4 Top ranking residues in 1j71A and their position on the structure 1 From SwissProt, id Q00663, 98% identical to 1j71A: 2.4.1 Clustering of residues at 25% coverage. 2 Description: Candidapepsin precursor (EC 3.4.23.24) (Aspartate 2.4.2 Overlap with known functional surfaces at ) (ACP). 25% coverage. 2 Organism, scientific name: Candida tropicalis (Yeast). 2.4.3 Possible novel functional surfaces at 25% Taxonomy: Eukaryota; Fungi; Ascomycota; Saccharomycotina; coverage. 5 Saccharomycetes; Saccharomycetales; mitosporic Saccharomyceta- les; Candida. 3 Notes on using trace results 7 Catalytic activity: Preferential cleavage at the carboxyl of hydro- 3.1 Coverage 7 phobic amino acids, but fails to cleave 15-Leu-—-Tyr-16, 16- 3.2 Known substitutions 7 Tyr-—-Leu-17 and 24-Phe-—-Phe-25 of B chain. Activates 3.3 Surface 7 , and degrades keratin. 3.4 Number of contacts 7 Subcellular location: Secreted. 3.5 Annotation 7 Ptm: O-glycosylated. 3.6 Mutation suggestions 7 Similarity: Belongs to the peptidase A1 family. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 7 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 7 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 8 no restrictions on its use as long as its content is in no way modified 4.3 Credits 8 and this statement is not removed.

1 Lichtarge lab 2006 Fig. 1. Residues 1-167 in 1j71A colored by their relative importance. (See Fig. 2. Residues 168-334 in 1j71A colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.) Appendix, Fig.9, for the coloring scheme.)

2.2 Multiple sequence alignment for 1j71A For the chain 1j71A, the alignment 1j71A.msf (attached) with 232 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 1j71A.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 232 Total number of residues: 72780 Smallest: 259 Largest: 334 Average length: 313.7 Alignment length: 334 Average identity: 31% Most related pair: 99% Most unrelated pair: 16% Most distant seq: 30%

Furthermore, <1% of residues show as conserved in this ali- Fig. 3. Residues in 1j71A, colored by their relative importance. Clockwise: gnment. front, back, top and bottom views. The alignment consists of 57% eukaryotic ( 14% vertebrata, <1% arthropoda, 40% fungi, <1% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the belong to. The clusters in Fig.4 are composed of the residues listed sequence descriptions can be found in the attachment, under the name in Table 1. 1j71A.descr. Table 1. 2.3 Residue ranking in 1j71A cluster size member color residues The 1j71A sequence is shown in Figs. 1–2, with each residue colored red 80 14,21,22,23,25,29,32,33,34 according to its estimated importance. The full listing of residues 35,36,37,38,39,40,65,70,80 in 1j71A can be found in the file called 1j71A.ranks sorted in the 82,84,85,86,92,94,97,109,111 attachment. 112,122,123,124,125,126,127 2.4 Top ranking residues in 1j71A and their position on 128,138,140,141,142,145,151 the structure 156,157,158,159,160,168,170 171,172,173,174,175,176,179 In the following we consider residues ranking among top 25% of resi- 180,182,184,216,217,218,219 dues in the protein . Figure 3 shows residues in 1j71A colored by their 220,221,222,298,299,300,301 importance: bright red and yellow indicate more conserved/important 302,303,304,305,307,308,310 residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. continued in next column 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the top 25% of all residues, this time colored according to clusters they

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) .(2) A(1)IER 298 I I(65) 0.17 2/0 3.65 L(18) V(10)A T(2)SMY FN 85 G G(60) 0.20 9/9 3.13 V(12) A(13) I(1) L(3)YFD E(1)KSQ TM

Table 2. The top 25% of residues in 1j71A at the interface with etha- nol.(Field names: res: residue number in the PDB entry; type: 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- Fig. 4. Residues in 1j71A, colored according to the cluster they belong to: ber of contacts realized through backbone atoms given in the bracket; dist: red, followed by blue and yellow are the largest clusters (see Appendix for distance of closest apporach to the ligand. ) the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 3. res type disruptive Table 1. continued mutations cluster size member 221 T (R)(K)(H)(FW) color residues 86 D (R)(FWH)(Y)(K) 311,312,319,321 298 I (R)(Y)(H)(K) 85 G (R)(K)(E)(H) Table 1. Clusters of top ranking residues in 1j71A. Table 3. List of disruptive mutations for the top 25% of residues in 1j71A, that are at the interface with ethanol. 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier Figure 5 shows residues in 1j71A colored by their importance, at the and the heteroatom name used in that file. interface with 1j71EOH575. Ethanol . Table 2 lists the top 25% of residues at Interface with the peptide 1j71B. Table 4 lists the top 25% the interface with 1j71EOH575 (ethanol). The following table (Table of residues at the interface with 1j71B. The following table (Table 3) suggests possible disruptive replacements for these residues (see 5) suggests possible disruptive replacements for these residues (see Section 3.6). Section 3.6). Table 2. Table 4. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) 221 T S(18) 0.13 2/0 4.45 34 G G(98)AN 0.01 17/17 3.44 T(73) S A(3) 35 S S(97)TE 0.02 5/2 3.41 G(1)QYV LV FKE 84 Y Y(97).F 0.03 28/17 3.34 86 D D(53) 0.15 7/6 3.86 LQ T(15) 218 D D(96).Q 0.03 11/0 2.69 S(22) GNHS G(1) 221 T S(18) 0.13 1/0 4.17 K(1) continued in next column continued in next column

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) V(15) T(2) F(3). M(3)S

Table 4. The top 25% of residues in 1j71A at the interface with 1j71B. (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 5. res type disruptive mutations 34 G (R)(KE)(H)(FW) 35 S (R)(K)(H)(FW) 84 Y (K)(Q)(M)(ER) Fig. 5. Residues in 1j71A, at the interface with ethanol, colored by their rela- 218 D (R)(FW)(H)(Y) tive importance. The ligand (ethanol) is colored green. Atoms further than 221 T (R)(K)(H)(FW) 30A˚ away from the geometric center of the ligand, as well as on the line of 82 I (R)(Y)(H)(T) sight to the ligand were removed. (See Appendix for the coloring scheme for 298 I (R)(Y)(H)(K) the protein chain 1j71A.) 85 G (R)(K)(E)(H) 216 V (R)(K)(Y)(E) Table 4. continued res type subst’s cvg noc/ dist Table 5. List of disruptive mutations for the top 25% of residues in 1j71A, that are at the interface with 1j71B. (%) bb (A˚ ) T(73) A(3) G(1)QYV FKE 82 I I(81) 0.15 5/0 4.00 L(8) V(2) T(1)M.F A(2)NED Y 298 I I(65) 0.17 4/0 3.94 L(18) V(10)A T(2)SMY FN 85 G G(60) 0.20 14/14 2.82 V(12) A(13) I(1) L(3)YFD E(1)KSQ TM 216 V L(36) 0.25 3/0 3.89 I(33) A(5) continued in next column Fig. 6. Residues in 1j71A, at the interface with 1j71B, colored by their rela- tive importance. 1j71B is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1j71A.)

4 Figure 6 shows residues in 1j71A colored by their importance, at the interface with 1j71B. Ethanol binding site. Table 6 lists the top 25% of residues at the interface with 1j71EOH573 (ethanol). 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˚ ) 184 L L(79) 0.09 2/0 4.35 I(11) V(1) F(4)MHY QE 312 D D(84) 0.09 4/0 3.73 N(10) R(1) .(2)LH 310 V V(85)L 0.11 8/0 3.55 .(1) F(1) E(4) Fig. 7. Residues in 1j71A, at the interface with ethanol, colored by their rela- I(3)T tive importance. The ligand (ethanol) is colored green. Atoms further than A(1)YS 30A˚ away from the geometric center of the ligand, as well as on the line of 319 S S(40) 0.19 5/0 3.57 sight to the ligand were removed. (See Appendix for the coloring scheme for G(44) the protein chain 1j71A.) .(3) A(3)F Q(2) shows (in blue) the rest of the larger cluster this surface belongs to. H(2) W(1)TMR

Table 6. The top 25% of residues in 1j71A at the interface with etha- nol.(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 Fig. 8. A possible active surface on the chain 1j71A. The larger cluster it 184 L (R)(Y)(T)(H) belongs to is shown in blue. 312 D (R)(FW)(H)(CG) 310 V (R)(K)(E)(Y) The residues belonging to this surface ”patch” are listed in Table 319 S (K)(R)(Q)(E) 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 1j71A, that are at the interface with ethanol. Table 8. res type substitutions(%) cvg 34 G G(98)ANS 0.01 Figure 7 shows residues in 1j71A colored by their importance, at the 220 G G(98)LKT 0.01 interface with 1j71EOH573. 32 D D(98).(1)T 0.02 2.4.3 Possible novel functional surfaces at 25% coverage. One 33 T T(98)SL 0.02 group of residues is conserved on the 1j71A surface, away from (or 35 S S(97)TELV 0.02 susbtantially larger than) other functional sites and interfaces reco- continued in next column gnizable in PDB entry 1j71. It is shown in Fig. 8. The right panel

5 Table 8. continued Table 8. continued res type substitutions(%) cvg res type substitutions(%) cvg 84 Y Y(97).FLQ 0.03 AT(2)SMYFN 92 G G(95)I(3).HF 0.03 159 Y Y(66)W(14)F(8)S 0.18 218 D D(96).QGNHS 0.03 D(2)A(2)INLCRHE 182 G G(94)P(1)D(1)QS 0.04 TK. 172 F F(87)L(9)I(1)Y 0.05 171 I L(46)A(1)I(26)N 0.19 168 G G(78)S(18)VCK. 0.06 M(5)T(10)SV(7)Y A(1) D 176 D D(90)N(8)VE 0.06 319 S S(40)G(44).(3) 0.19 14 Y Y(95).(2)LSFI 0.07 A(3)FQ(2)H(2) 122 G G(93)P(2)A(2)NS 0.07 W(1)TMR Q 85 G G(60)V(12)A(13) 0.20 160 L L(88)SVM(5)F(2) 0.08 I(1)L(3)YFDE(1) YAI. KSQTM 305 R R(75)K(8)G(7) 0.08 151 I I(65)L(11)W(1) 0.21 N(1)EHT(2)S(1)A V(14)T(6)F.CM VQ 109 Q V(4)Q(50)M(2) 0.22 184 L L(79)I(11)V(1) 0.09 L(22)F(12)A(3)T F(4)MHYQE .I(1)NYGCK 312 D D(84)N(10)R(1) 0.09 123 I L(9)I(60)V(25) 0.22 .(2)LH T(2)GYMF 222 T T(46)S(47)VDGNY 0.10 124 M L(72)M(10)V(2) 0.23 I(1)LPQ F(12)GAIE 38 L F(3)L(84)I(1) 0.11 158 L I(8)F(22)L(45) 0.23 T(5)A(1)M(2) V(20)CM(2). V(1) 175 V V(47)L(1)I(33) 0.23 310 V V(85)L.(1)F(1) 0.11 K(2)Y(6)S(6)TAF E(4)I(3)TA(1)YS NM 127 G G(69)A(19)S(9)N 0.12 180 Y Y(74)I(3)F(18)E 0.23 VP VKH 140 N N(59)T(6)P(27)Y 0.12 302 N D(1)V(27)N(18) 0.24 S(2)A(1)L(1)GMV A(11)T(25)S(9)M 219 S T(51)S(45)P(1)A 0.13 PLKI(2)F VY 128 F L(18)Y(43)W(1) 0.25 221 T S(18)T(73)A(3) 0.13 R(1)F(25)HP(4) G(1)QYVFKE V(1)IKMGA 80 F F(72)V(3)Y(4)N 0.14 161 N N(46)S(13)A(2) 0.25 L(7)W(3)DRK.A P(1)T(1)QG(15) I(1)TG D(12)R(1)K(3) 321 A A(88).(4)SG(4) 0.14 E(1). T(1) 216 V L(36)I(33)A(5) 0.25 82 I I(81)L(8)V(2) 0.15 V(15)T(2)F(3). T(1)M.FA(2)NEDY M(3)S 86 D D(53)T(15)S(22) 0.15 G(1)K(1).(2) Table 8. Residues forming surface ”patch” in 1j71A. A(1)IER 138 Y Y(58).(7)A(6) 0.16 I(8)V(8)K(1)N Table 9. F(3)QS(1)LDTR res type disruptive 179 K R(3)Y(13)K(62)M 0.17 mutations H(10)L(4)FQ(1) 34 G (R)(KE)(H)(FW) A(1)C 220 G (R)(E)(FKWH)(YD) 298 I I(65)L(18)V(10) 0.17 32 D (R)(FWH)(K)(VA) continued in next column 33 T (R)(K)(H)(FQW) 35 S (R)(K)(H)(FW) continued in next column

6 Table 9. continued evolutionary pressure. (I.e., the smaller the coverage, the stronger the res type disruptive pressure on the residue.) Starting from the top of that list, mutating a mutations couple of residues should affect the protein somehow, with the exact 84 Y (K)(Q)(M)(ER) effects to be determined experimentally. 92 G (E)(K)(R)(Q) 218 D (R)(FW)(H)(Y) 3.2 Known substitutions 182 G (R)(K)(H)(FW) One of the table columns is “substitutions” - other amino acid types 172 F (K)(E)(TQR)(D) seen at the same position in the alignment. These amino acid types 168 G (ER)(K)(H)(FW) may be interchangeable at that position in the protein, so if one wants 176 D (R)(H)(FW)(Y) to affect the protein by a point mutation, they should be avoided. For 14 Y (K)(Q)(R)(EM) example if the substitutions are “RVK” and the original protein has 122 G (R)(E)(KH)(FYW) an R at that position, it is advisable to try anything, but RVK. Conver- 160 L (R)(Y)(H)(K) sely, when looking for substitutions which will not affect the protein, 305 R (Y)(D)(T)(E) one may try replacing, R with K, or (perhaps more surprisingly), with 184 L (R)(Y)(T)(H) V. The percentage of times the substitution appears in the alignment 312 D (R)(FW)(H)(CG) is given in the immediately following bracket. No percentage is given 222 T (R)(K)(H)(FW) in the cases when it is smaller than 1%. This is meant to be a rough 38 L (R)(Y)(H)(K) guide - due to rounding errors these percentages often do not add up 310 V (R)(K)(E)(Y) to 100%. 127 G (R)(KE)(H)(Y) 140 N (Y)(H)(R)(FW) 3.3 Surface 219 S (KR)(Q)(H)(EM) To detect candidates for novel functional interfaces, first we look for 221 T (R)(K)(H)(FW) residues that are solvent accessible (according to DSSP program) by 2 80 F (E)(K)(D)(Q) at least 10A˚ , which is roughly the area needed for one water mole- 321 A (KR)(E)(YQ)(H) cule to come in the contact with the residue. Furthermore, we require 82 I (R)(Y)(H)(T) that these residues form a “cluster” of residues which have neighbor 86 D (R)(FWH)(Y)(K) within 5A˚ from any of their heavy atoms. 138 Y (K)(QR)(M)(E) Note, however, that, if our picture of protein evolution is correct, 179 K (Y)(T)(FW)(S) the neighboring residues which are not surface accessible might be 298 I (R)(Y)(H)(K) equally important in maintaining the interaction specificity - they 159 Y (K)(Q)(M)(R) should not be automatically dropped from consideration when choo- 171 I (R)(Y)(H)(K) sing the set for mutagenesis. (Especially if they form a cluster with 319 S (K)(R)(Q)(E) the surface residues.) 85 G (R)(K)(E)(H) 151 I (R)(Y)(H)(K) 3.4 Number of contacts 109 Q (Y)(H)(FW)(T) Another column worth noting is denoted “noc/bb”; it tells the num- 123 I (R)(Y)(KH)(E) ber of contacts heavy atoms of the residue in question make across 124 M (Y)(H)(R)(T) the interface, as well as how many of them are realized through the 158 L (R)(Y)(H)(T) backbone atoms (if all or most contacts are through the backbone, 175 V (R)(Y)(K)(E) mutation presumably won’t have strong impact). Two heavy atoms 180 Y (K)(Q)(R)(M) are considered to be “in contact” if their centers are closer than 5A˚ . 302 N (Y)(H)(R)(T) 128 F (E)(K)(D)(T) 3.5 Annotation 161 N (Y)(FWH)(T)(R) If the residue annotation is available (either from the pdb file or 216 V (R)(K)(Y)(E) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 9. Disruptive mutations for the surface patch in 1j71A. (indicating existence of related site record in PDB ), S-S (disulfide 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 ],

7 bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated COVERAGE evolutionary pressure they experience can be seen in Fig. 9. 4.3 Credits V 4.3.1 Alistat alistat reads a multiple sequence alignment from the 100% 50% 30% 5% file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi-

V ned as (idents / MIN(len1, len2)) where idents is the number of RELATIVE IMPORTANCE exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, Fig. 9. Coloring scheme used to color residues by their relative importance. 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 and NH2 group possession [NQRK]. The suggestions are listed numbers (hence, the most outlying sequence). alistat is copyrighted according to how different they appear to be from the original amino by HHMI/Washington University School of Medicine, 1992-2001, acid, and they are grouped in round brackets if they appear equally and freely distributed under the GNU General Public License. disruptive. From left to right, each bracketed group of amino acid 4.3.2 CE To map ligand binding sites from different types resembles more strongly the original (i.e. is, presumably, less source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 4.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 4 APPENDIX 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 4.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: 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

8 users: the attached package needs to be unzipped for Pymol to read is copyrighted by Lichtarge Lab, Baylor College of Medicine, the scripts and launch the viewer.) Houston. 4.4 Note about ET Viewer 4.7 Attachments Dan Morgan from the Lichtarge lab has developed a visualization The following files should accompany this report: tool specifically for viewing trace results. If you are interested, please • 1j71A.complex.pdb - coordinates of 1j71A with all of its inter- visit: acting partners http://mammoth.bcm.tmc.edu/traceview/ • 1j71A.etvx - ET viewer input file for 1j71A The viewer is self-unpacking and self-installing. Input files to be used • 1j71A.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 1j71A main report. • 1j71A.ranks - Ranks file in sequence order for 1j71A 4.5 Citing this work • 1j71A.clusters - Cluster descriptions for 1j71A The method used to rank residues and make predictions in this report • 1j71A.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 1j71A Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1j71A.descr - description of sequences used in 1j71A msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1j71A.ranks sorted - full listing of residues and their ranking for of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 1j71A tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 1j71A.1j71EOH575.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 1j71A.cbcvg - used by other 1j71A – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1j71A.1j71B.if.pml - Pymol script for Figure 6 of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 1j71A.1j71EOH573.if.pml - Pymol script for Figure 7 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker

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