Pages 1–9 1n47 Evolutionary trace report by report maker June 16, 2009

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

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1n47): Title: Isolectin b4 from villosa in complex with the tn antigen Compound: Mol id: 1; molecule: isolectin b4; chain: a, b, c, d CONTENTS Organism, scientific name: Vicia Villosa; 1n47 contains a single unique chain 1n47A (233 residues long) and 1 Introduction 1 its homologues 1n47D, 1n47C, and 1n47B. 2 Chain 1n47A 1 2.1 Q9ZWP5 overview 1 2.2 Multiple sequence alignment for 1n47A 1 2.3 Residue ranking in 1n47A 1 2.4 Top ranking residues in 1n47A and their position on 2 CHAIN 1N47A the structure 2 2.1 Q9ZWP5 overview 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at From SwissProt, id Q9ZWP5, 54% identical to 1n47A: 25% coverage. 2 Description: Lectin. 2.4.3 Possible novel functional surfaces at 25% Organism, scientific name: Robinia pseudoacacia (Black locust). coverage. 5 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3 Notes on using trace results 7 eudicotyledons; ; eurosids I; ; ; Papilionoi- 3.1 Coverage 7 deae; Robinieae; Robinia. 3.2 Known substitutions 7 3.3 Surface 8 3.4 Number of contacts 8 3.5 Annotation 8 2.2 Multiple sequence alignment for 1n47A 3.6 Mutation suggestions 8 For the chain 1n47A, the alignment 1n47A.msf (attached) with 331 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 8 database, and fragments shorter than 75% of the query as well as 4.1 File formats 8 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 8 to this report, under the name of 1n47A.msf. Its statistics, from the 4.3 Credits 8 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-116 in 1n47A colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.)

Fig. 2. Residues 117-233 in 1n47A colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.)

Fig. 3. Residues in 1n47A, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 331 Total number of residues: 73572 Smallest: 175 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 233 Average length: 222.3 Alignment length: 233 Average identity: 37% Most related pair: 99% Most unrelated pair: 12% Most distant seq: 33%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 60% eukaryotic ( 60% plantae), and <1% 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 1n47A.descr. 2.3 Residue ranking in 1n47A The 1n47A sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1n47A can be found in the file called 1n47A.ranks sorted in the attachment. 2.4 Top ranking residues in 1n47A and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 1n47A, colored according to the cluster they belong to: residues in the protein . Figure 3 shows residues in 1n47A colored red, followed by blue and yellow are the largest clusters (see Appendix for by their importance: bright red and yellow indicate more conser- the coloring scheme). Clockwise: front, back, top and bottom views. The ved/important residues (see Appendix for the coloring scheme). A corresponding Pymol script is attached. Pymol script for producing this figure can be found in the attachment. in Table 1. 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 1. cluster size member color residues red 54 6,11,32,34,35,48,49,50,51,52 65,66,67,69,71,84,85,86,89 90,91,105,106,107,108,120 121,122,123,124,125,126,129 132,137,138,139,140,141,143 147,165,167,174,195,206,207 208,211,212,226,227,228,232 blue 2 55,203

Table 1. Clusters of top ranking residues in 1n47A.

2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Interface with 1n47B.Table 2 lists the top 25% of residues at the interface with 1n47B. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. Fig. 5. Residues in 1n47A, at the interface with 1n47B, colored by their rela- res type subst’s cvg noc/ dist tive importance. 1n47B is shown in backbone representation (See Appendix (%) bb (A˚ ) for the coloring scheme for the protein chain 1n47A.) 55 P P(86) 0.14 25/7 3.57 R(1) Q(4)K Figure 5 shows residues in 1n47A colored by their importance, at the A(3) interface with 1n47B. L(1)TYG Manganese (ii) ion binding site. Table 4 lists the top 25% of resi- S. dues at the interface with 1n47AMN253 (manganese (ii) ion). The 6 F F(90)ST 0.15 13/9 3.37 following table (Table 5) suggests possible disruptive replacements L(1) for these residues (see Section 3.6). I(1) Table 4. .(2)DA res type subst’s cvg noc/ dist Y(1)VW (%) bb (A˚ ) 52 Y Y(82) 0.18 10/0 3.67 125 D D(92) 0.03 4/0 1.93 H(8) N(3)VGF F(6)CLS L(1)TIK AWG. . 123 E E(87) 0.04 4/0 2.32 Table 2. The top 25% of residues in 1n47A at the interface with 1n47B. V(7) (Field names: res: residue number in the PDB entry; type: amino acid type; A(3)DWC substs: substitutions seen in the alignment; with the percentage of each type KQ. 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 137 H H(84) 0.04 5/0 2.49 of closest apporach to the ligand. ) R(4) S(3) .(1)LAE Table 3. D(1) res type disruptive Q(1)IPG mutations 147 S S(88) 0.06 1/0 4.29 55 P (Y)(R)(H)(T) P(7)DHW 6 F (K)(E)(Q)(R) IV.KNEF 52 Y (K)(Q)(E)(M) A 132 D D(81) 0.07 5/1 2.39 Table 3. List of disruptive mutations for the top 25% of residues in continued in next column 1n47A, that are at the interface with 1n47B.

3 Table 4. continued Figure 6 shows residues in 1n47A colored by their importance, at the res type subst’s cvg noc/ dist interface with 1n47AMN253. (%) bb (A˚ ) Calcium ion binding site. Table 6 lists the top 25% of residues at .(9) the interface with 1n47ACA252 (calcium ion). The following table E(4) (Table 7) suggests possible disruptive replacements for these residues S(2)YNH (see Section 3.6). LKR Table 6. res type subst’s cvg noc/ dist Table 4. The top 25% of residues in 1n47A at the interface with manga- A˚ nese (ii) ion.(Field names: res: residue number in the PDB entry; type: amino (%) bb ( ) acid type; substs: substitutions seen in the alignment; with the percentage of 125 D D(92) 0.03 4/0 2.43 each type in the bracket; noc/bb: number of contacts with the ligand, with N(3)VGF the number of contacts realized through backbone atoms given in the bracket; L(1)TIK dist: distance of closest apporach to the ligand. ) . 137 H H(84) 0.04 1/0 4.78 R(4) S(3) Table 5. .(1)LAE res type disruptive D(1) mutations Q(1)IPG 125 D (R)(H)(FW)(Y) 132 D D(81) 0.07 4/0 2.25 123 E (H)(FW)(Y)(R) .(9) 137 H (E)(T)(QD)(M) E(4) 147 S (R)(K)(H)(Q) S(2)YNH 132 D (R)(FW)(H)(YVCAG) LKR 85 D D(59) 0.16 2/1 4.63 Y(10) Table 5. List of disruptive mutations for the top 25% of residues in 1n47A, that are at the interface with manganese (ii) ion. H(12) E(4) V(2)S N(1)T A(1)P F(2) G(2)IR. Q 129 N N(73) 0.20 6/2 2.71 A(2) D(8) S(7) G(2)R Y(3) Q(1)KET H

Table 6. The top 25% of residues in 1n47A 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. )

Table 7. res type disruptive mutations Fig. 6. Residues in 1n47A, at the interface with manganese (ii) ion, colored 125 D (R)(H)(FW)(Y) by their relative importance. The ligand (manganese (ii) ion) is colored green. 137 H (E)(T)(QD)(M) A Atoms further than 30 ˚ away from the geometric center of the ligand, as well continued in next column as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1n47A.)

4 Table 7. continued Table 8. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 132 D (R)(FW)(H)(YVCAG) S(23)L. 85 D (R)(H)(FW)(K) GV(1) 129 N (Y)(FW)(H)(T) N(2)RAI C Table 7. List of disruptive mutations for the top 25% of residues in 85 D D(59) 0.16 16/0 2.72 1n47A, that are at the interface with calcium ion. Y(10) H(12) E(4) V(2)S N(1)T A(1)P F(2) G(2)IR. Q 129 N N(73) 0.20 19/1 2.90 A(2) D(8) S(7) G(2)R Y(3) Q(1)KET H 212 G G(83) 0.21 9/9 3.76 A(3) E(1) T(2). S(3) Q(1) V(1)LKR YP

Fig. 7. Residues in 1n47A, at the interface with calcium ion, colored by their Table 8. The top 25% of residues in 1n47A at the interface with relative importance. The ligand (calcium ion) is colored green. Atoms further TNR.(Field names: res: residue number in the PDB entry; type: amino acid A˚ than 30 away from the geometric center of the ligand, as well as on the line type; substs: substitutions seen in the alignment; with the percentage of each of sight to the ligand were removed. (See Appendix for the coloring scheme type in the bracket; noc/bb: number of contacts with the ligand, with the num- for the protein chain 1n47A.) ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Figure 7 shows residues in 1n47A colored by their importance, at the interface with 1n47ACA252. TNR binding site. Table 8 lists the top 25% of residues at the inter- Table 9. face with 1n47TNR1401 (tnr). The following table (Table 9) suggests res type disruptive possible disruptive replacements for these residues (see Section 3.6). mutations 84 A (R)(K)(Y)(E) Table 8. 211 T (R)(K)(H)(FW) res type subst’s cvg noc/ dist 85 D (R)(H)(FW)(K) (%) bb (A˚ ) 129 N (Y)(FW)(H)(T) 84 A A(49) 0.14 3/0 4.70 212 G (R)(KE)(H)(FW) G(38) S(2)L Table 9. List of disruptive mutations for the top 25% of residues in P(2) 1n47A, that are at the interface with TNR. V(1)D T(2)N.C 211 T T(68) 0.15 1/1 4.66 Figure 8 shows residues in 1n47A colored by their importance, at the interface with 1n47TNR1401. continued in next column 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1n47A surface, away from (or

5 Table 10. continued res type substitutions(%) cvg 122 V V(93)I(3)NL(1)C 0.05 YW. 147 S S(88)P(7)DHWIV. 0.06 KNEFA 167 Y Y(87)QF(9)SV.TL 0.07 I 226 W W(95).(1)GRPLSF 0.08 66 S S(77)D(9)N(9)TA 0.09 PCHG. 195 L L(87)V(5)I(1) 0.11 .(1)F(1)PM(1)S 165 I I(76)V(19)PN 0.12 L(1)WA.MFT 55 P P(86)R(1)Q(4)K 0.14 A(3)L(1)TYGS. 6 F F(90)STL(1)I(1) 0.15 .(2)DAY(1)VW 140 I I(78)F(1)V(11) 0.17 L(5)GKSNA 52 Y Y(82)H(8)F(6)CL 0.18 Fig. 8. Residues in 1n47A, at the interface with TNR, colored by their relative SAWG. importance. The ligand (TNR) is colored green. Atoms further than 30A˚ away 91 L L(62)I(25)M(2) 0.18 from the geometric center of the ligand, as well as on the line of sight to the V(7)F.N ligand were removed. (See Appendix for the coloring scheme for the protein 11 F F(83)H(2)S(2)V 0.19 chain 1n47A.) .(1)PL(2)GD(2)K WI(1)RA(1)M 93 P P(78)R(1)S(8) 0.20 susbtantially larger than) other functional sites and interfaces reco- G(4)A(4)N(1)KHT gnizable in PDB entry 1n47. It is shown in Fig. 9. The right panel 120 V V(67)L(11)F(12) 0.22 shows (in blue) the rest of the larger cluster this surface belongs to. I(6)EQA. 227 S S(75).(2)E(4) 0.23 F(1)T(3)R(1) N(3)Y(6)HKGA 232 L L(56)F(11).(28) 0.23 V(1)M(1)I(1)T 51 L F(43)L(33)I(3) 0.24 T(4)M(1)S(7)R V(1)GW(1)A(1). 174 L L(81)I(7)V(3) 0.24 M(3)F(2)QG.SEDA

Table 10. Residues forming surface ”patch” in 1n47A.

Fig. 9. A possible active surface on the chain 1n47A. The larger cluster it belongs to is shown in blue. Table 11. res type disruptive The residues belonging to this surface ”patch” are listed in Table 10, mutations while Table 11 suggests possible disruptive replacements for these 67 F (KE)(T)(D)(Q) residues (see Section 3.6). 71 F (K)(E)(Q)(T) 89 F (K)(E)(Q)(R) Table 10. 122 V (R)(KE)(Y)(QHD) res type substitutions(%) cvg 147 S (R)(K)(H)(Q) 67 F F(95)W(2)SQL. 0.01 167 Y (K)(QR)(EM)(N) 71 F F(97)IH.VLY 0.01 226 W (E)(K)(D)(TQ) 89 F F(97)L(1)SC. 0.02 continued in next column continued in next column

6 Table 11. continued Table 12. continued res type disruptive res type substitutions(%) cvg mutations 107 L L(74)I(7)V(10) 0.17 66 S (R)(K)(H)(Q) .(3)DYM(1)QT(1) 195 L (R)(Y)(H)(T) FHS 165 I (R)(Y)(H)(T) 129 N N(73)A(2)D(8) 0.20 55 P (Y)(R)(H)(T) S(7)G(2)RY(3) 6 F (K)(E)(Q)(R) Q(1)KETH 140 I (Y)(R)(H)(E) 212 G G(83)A(3)E(1) 0.21 52 Y (K)(Q)(E)(M) T(2).S(3)Q(1) 91 L (Y)(R)(T)(H) V(1)LKRYP 11 F (E)(K)(T)(D) 93 P (Y)(R)(H)(E) Table 12. Residues forming surface ”patch” in 1n47A. 120 V (Y)(R)(K)(E) 227 S (K)(R)(M)(FQW) 232 L (R)(Y)(H)(T) 51 L (R)(Y)(H)(KE) Table 13. 174 L (R)(Y)(H)(T) res type disruptive mutations Table 11. Disruptive mutations for the surface patch in 1n47A. 132 D (R)(FW)(H)(YVCAG) 126 T (R)(K)(H)(Q) 105 L (R)(Y)(T)(H) Another group of surface residues is shown in Fig.10. The right panel 85 D (R)(H)(FW)(K) shows (in blue) the rest of the larger cluster this surface belongs to. 107 L (R)(Y)(H)(T) 129 N (Y)(FW)(H)(T) 212 G (R)(KE)(H)(FW)

Table 13. Disruptive mutations for the surface patch in 1n47A.

3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to some small top percentage of residues [100% is all of the residues Fig. 10. Another possible active surface on the chain 1n47A. The larger in a chain], according to trace. The ET results are presented in the cluster it belongs to is shown in blue. form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed The residues belonging to this surface ”patch” are listed in Table 12, evolutionary pressure. (I.e., the smaller the coverage, the stronger the while Table 13 suggests possible disruptive replacements for these pressure on the residue.) Starting from the top of that list, mutating a residues (see Section 3.6). couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. Table 12. res type substitutions(%) cvg 3.2 Known substitutions 132 D D(81).(9)E(4) 0.07 One of the table columns is “substitutions” - other amino acid types S(2)YNHLKR seen at the same position in the alignment. These amino acid types 126 T T(85)S(6)N(4)VI 0.09 may be interchangeable at that position in the protein, so if one wants GLAP. to affect the protein by a point mutation, they should be avoided. For 105 L L(85)F(4)I(1)H 0.11 example if the substitutions are “RVK” and the original protein has .(3)G(1)RTYMVQ an R at that position, it is advisable to try anything, but RVK. Conver- 85 D D(59)Y(10)H(12) 0.16 sely, when looking for substitutions which will not affect the protein, E(4)V(2)SN(1)T one may try replacing, R with K, or (perhaps more surprisingly), with A(1)PF(2)G(2)IR V. The percentage of times the substitution appears in the alignment .Q is given in the immediately following bracket. No percentage is given continued in next column in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up to 100%.

7 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 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 COVERAGE that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. V Note, however, that, if our picture of protein evolution is correct, 100% 50% 30% 5% the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) V

3.4 Number of contacts RELATIVE IMPORTANCE 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 Fig. 11. Coloring scheme used to color residues by their 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˚ . • variability has two subfields: 1. number of different amino acids appearing in in this column 3.5 Annotation of the alignment If the residue annotation is available (either from the pdb file or 2. their type from other sources), another column, with the header “annotation” • rho ET score - the smaller this value, the lesser variability of appears. Annotations carried over from PDB are the following: site this position across the branches of the tree (and, presumably, (indicating existence of related site record in PDB ), S-S (disulfide the greater the importance for the protein) bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 3.6 Mutation suggestions • gaps percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein The following color scheme is used in figures with residues colored with its ligand. The attempt is made to complement the following by cluster size: black is a single-residue cluster; clusters composed of properties: small [AV GSTC], medium [LPNQDEMIK], large more than one residue colored according to this hierarchy (ordered [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by descending size): red, blue, yellow, green, purple, azure, tur- tively [KHR], or negatively [DE] charged, aromatic [WFYH], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, long aliphatic chain [EKRQM], OH-group possession [SDETY ], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, and NH2 group possession [NQRK]. The suggestions are listed DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, according to how different they appear to be from the original amino tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. acid, and they are grouped in round brackets if they appear equally The colors used to distinguish the residues by the estimated disruptive. From left to right, each bracketed group of amino acid evolutionary pressure they experience can be seen in Fig. 11. types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 4 APPENDIX alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of Files with extension “ranks sorted” are the actual trace results. The exact identities and len1, len2 are the unaligned lengths of the two fields in the table in this file: sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • residue# residue number in the PDB file seq” is calculated by finding the maximum pairwise identity (best • type amino acid type relative) for all N sequences, then finding the minimum of these N • rank rank of the position according to older version of ET numbers (hence, the most outlying sequence). alistat is copyrighted

8 by HHMI/Washington University School of Medicine, 1992-2001, The viewer is self-unpacking and self-installing. Input files to be used and freely distributed under the GNU General Public License. with ETV (extension .etvx) can be found in the attachment to the main report. 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: 4.5 Citing this work http://cl.sdsc.edu/ . Shindyalov IN, Bourne PE (1998) The method used to rank residues and make predictions in this report ”Protein structure alignment by incremental combinatorial extension can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of (CE) of the optimal path . Protein Engineering 11(9) 739-747. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 4.3.3 DSSP In this work a residue is considered solvent accessi- by Importance” J. Mol. Bio. 336: 1265-82. For the original version ble if the DSSP program finds it exposed to water by at least 10A˚ 2, of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- which is roughly the area needed for one water molecule to come in tionary Trace Method Defines Binding Surfaces Common to Protein the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Families” J. Mol. Bio. 257: 342-358. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version report maker itself is described in Mihalek I., I. Res and O. by [email protected] November 18,2002, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics http://www.cmbi.kun.nl/gv/dssp/descrip.html. 22:1656-7. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 4.6 About report maker gnment as a starting point for the analysis (sequences shorter than report maker was written in 2006 by Ivana Mihalek. The 1D ran- 75% of the query are taken out, however); R. Schneider, A. de king visualization program was written by Ivica Res.ˇ report maker Daruvar, and C. Sander. ”The HSSP database of protein structure- is copyrighted by Lichtarge Lab, Baylor College of Medicine, sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. Houston. http://swift.cmbi.kun.nl/swift/hssp/ 4.7 Attachments A 4.3.5 LaTex The text for this report was processed using LTEX; The following files should accompany this report: Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • 1n47A.complex.pdb - coordinates of 1n47A with all of its interacting partners 4.3.6 Muscle When making alignments “from scratch”, report • maker uses Muscle alignment program: Edgar, Robert C. (2004), 1n47A.etvx - ET viewer input file for 1n47A ”MUSCLE: multiple sequence alignment with high accuracy and • 1n47A.cluster report.summary - Cluster report summary for high throughput.” Nucleic Acids Research 32(5), 1792-97. 1n47A • http://www.drive5.com/muscle/ 1n47A.ranks - Ranks file in sequence order for 1n47A • 1n47A.clusters - Cluster descriptions for 1n47A 4.3.7 Pymol The figures in this report were produced using • Pymol. The scripts can be found in the attachment. Pymol 1n47A.msf - the multiple sequence alignment used for the chain is an open-source application copyrighted by DeLano Scien- 1n47A tific LLC (2005). For more information about Pymol see • 1n47A.descr - description of sequences used in 1n47A msf http://pymol.sourceforge.net/. (Note for Windows • 1n47A.ranks sorted - full listing of residues and their ranking users: the attached package needs to be unzipped for Pymol to read for 1n47A the scripts and launch the viewer.) • 1n47A.1n47B.if.pml - Pymol script for Figure 5 4.4 Note about ET Viewer • 1n47A.cbcvg - used by other 1n47A – related pymol scripts Dan Morgan from the Lichtarge lab has developed a visualization • 1n47A.1n47AMN253.if.pml - Pymol script for Figure 6 tool specifically for viewing trace results. If you are interested, please • 1n47A.1n47ACA252.if.pml - Pymol script for Figure 7 visit: • 1n47A.1n47TNR1401.if.pml - Pymol script for Figure 8 http://mammoth.bcm.tmc.edu/traceview/

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