Pages 1–8 1civ Evolutionary trace report by report maker August 2, 2010

4.3.1 Alistat 7 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 8

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1civ): Title: Chloroplast nadp-dependent malate dehydrogenase from fla- veria bidentis Compound: Mol id: 1; molecule: nadp-malate dehydrogenase; CONTENTS chain: a; ec: 1.1.1.82 Organism, scientific name: Bidentis; 1 Introduction 1 1civ contains a single unique chain 1civA (374 residues long). 2 Chain 1civA 1 2.1 Q42737 overview 1 2.2 Multiple sequence alignment for 1civA 1 2.3 Residue ranking in 1civA 1 2 CHAIN 1CIVA 2.4 Top ranking residues in 1civA and their position on the structure 1 2.1 Q42737 overview 2.4.1 Clustering of residues at 25% coverage. 2 From SwissProt, id Q42737, 100% identical to 1civA: 2.4.2 Overlap with known functional surfaces at Description: NADP-malate dehydrogenase. 25% coverage. 2 Organism, scientific name: Flaveria trinervia (Clustered yellow- 2.4.3 Possible novel functional surfaces at 25% tops). coverage. 4 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3 Notes on using trace results 6 eudicotyledons; ; campanulids; ; ; Aste- 3.1 Coverage 6 roideae; Tageteae; Flaveria. 3.2 Known substitutions 6 3.3 Surface 7 3.4 Number of contacts 7 3.5 Annotation 7 2.2 Multiple sequence alignment for 1civA 3.6 Mutation suggestions 7 For the chain 1civA, the alignment 1civA.msf (attached) with 308 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 7 database, and fragments shorter than 75% of the query as well as 4.1 File formats 7 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 7 to this report, under the name of 1civA.msf. Its statistics, from the 4.3 Credits 7 alistat program are the following:

1 Lichtarge lab 2006 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.

Fig. 1. Residues 12-198 in 1civA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 2. Residues 199-385 in 1civA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.) Fig. 3. Residues in 1civA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 308 Total number of residues: 100746 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 284 top 25% of all residues, this time colored according to clusters they Largest: 374 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 327.1 in Table 1. Alignment length: 374 Average identity: 50% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 24% color residues Most distant seq: 38% red 72 49,52,54,56,58,116,124,126 133,134,135,136,138,139,140 143,146,147,150,151,154,158 Furthermore, <1% of residues show as conserved in this ali- 166,167,168,169,170,171,172 gnment. 173,174,175,182,189,190,193 The alignment consists of 29% eukaryotic ( 4% vertebrata, <1% 195,196,197,199,200,201,222 arthropoda, 14% plantae), and 10% prokaryotic sequences. (Des- 223,224,225,226,229,231,232 criptions of some sequences were not readily available.) The file 257,261,264,265,268,269,272 containing the sequence descriptions can be found in the attachment, 273,275,276,277,279,280,281 under the name 1civA.descr. 283,302,312,321,323,354,355 2.3 Residue ranking in 1civA 358 blue 11 76,78,88,90,93,94,95,97,99 The 1civA sequence is shown in Figs. 1–2, with each residue colored 101,103 according to its estimated importance. The full listing of residues yellow 3 63,65,69 in 1civA can be found in the file called 1civA.ranks sorted in the green 2 291,330 attachment. purple 2 129,130 2.4 Top ranking residues in 1civA and their position on azure 2 294,295 the structure continued in next column In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1civA colored by their

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) VS 272 W R(85) 0.07 1/1 4.77 W(13)S. 99 L A(83) 0.11 70/24 3.55 L(13) C(1). S(1)T 275 S S(94)YA 0.11 4/4 3.90 PT(3)G 95 L L(92) 0.13 2/2 4.79 I(5).M 93 M M(92) 0.16 44/10 3.32 A(2)Y. L(2)FI 88 L L(84) 0.17 2/2 4.53 V(5) A(7) M(1).QC 101 P P(95)GR 0.17 5/5 4.49 Fig. 4. Residues in 1civA, colored according to the cluster they belong to: TE.DLAQ red, followed by blue and yellow are the largest clusters (see Appendix for 103 L L(92) 0.19 12/9 3.29 the coloring scheme). Clockwise: front, back, top and bottom views. The V(5)IF. corresponding Pymol script is attached. C 268 L I(59) 0.20 28/4 3.45 L(13) Table 1. continued V(27). cluster size member 52 G G(93) 0.21 1/1 5.00 color residues .(5)V 206 A A(85) 0.22 4/2 3.71 Table 1. Clusters of top ranking residues in 1civA. S(13)GL 56 N Y(80) 0.23 26/6 3.13 N(13) 2.4.2 Overlap with known functional surfaces at 25% coverage. .(4) The name of the ligand is composed of the source PDB identifier S(1) and the heteroatom name used in that file. 91 V T(4) 0.25 10/6 3.40 Interface with 1civA1.Table 2 lists the top 25% of residues at V(82) the interface with 1civA1. The following table (Table 3) suggests L(7) possible disruptive replacements for these residues (see Section 3.6). I(2) Table 2. C(2). res type subst’s cvg noc/ dist 278 A A(69) 0.25 26/14 3.49 (%) bb (A˚ ) L(13)I 264 R R(99). 0.02 54/1 2.72 M(13) 97 D D(99). 0.03 63/14 2.72 G(1)FVS 200 R R(99)K 0.03 22/10 3.77 94 E E(99)Q. 0.04 61/11 2.70 Table 2. The top 25% of residues in 1civA at the interface with 1civA1. 279 S S(99)EC 0.04 28/8 2.70 (Field names: res: residue number in the PDB entry; type: amino acid type; 90 G G(94) 0.06 13/13 3.45 substs: substitutions seen in the alignment; with the percentage of each type A(5). in the bracket; noc/bb: number of contacts with the ligand, with the number of 199 N N(98)LD 0.06 28/15 3.52 contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) 276 S S(98)AR 0.06 9/7 3.25 P 277 A A(96)ET 0.06 4/4 3.85 continued in next column

3 Table 3. Figure 5 shows residues in 1civA colored by their importance, at the res type disruptive interface with 1civA1. mutations NAP binding site. Table 4 lists the top 25% of residues at the inter- 264 R (TD)(SVCLAPIG)(YE)(FMW) face with 1civNAP386 (nap). The following table (Table 5) suggests 97 D (R)(FWH)(VCAG)(KY) possible disruptive replacements for these residues (see Section 3.6). 200 R (T)(YD)(SVCAG)(FELWPI) 94 E (FWH)(YVCAG)(TR)(S) Table 4. 279 S (R)(K)(FWH)(Q) res type subst’s cvg noc/ dist 90 G (KER)(HD)(Q)(FMW) (%) bb (A˚ ) 199 N (Y)(H)(FW)(TR) 143 N N(99)H 0.02 1/0 4.51 276 S (KR)(YH)(FEQW)(M) 196 L L(99)I 0.02 6/0 3.88 277 A (R)(K)(Y)(H) 197 D D(99)E 0.02 1/0 4.79 272 W (E)(K)(D)(TQ) 169 N N(99)GD 0.04 54/18 2.69 99 L (R)(Y)(H)(K) M 275 S (KR)(Q)(H)(M) 146 I I(98)LV 0.05 14/0 3.85 95 L (Y)(R)(TH)(SCG) 276 S S(98)AR 0.06 1/0 4.90 93 M (Y)(T)(HR)(CG) P 88 L (Y)(R)(H)(T) 126 G G(92) 0.07 31/31 3.85 101 P (Y)(R)(H)(T) A(5)SDC 103 L (R)(Y)(H)(T) 225 H AH(99)P 0.07 21/0 2.90 268 L (YR)(H)(T)(KE) R 52 G (KER)(HD)(Q)(FMW) 168 G G(84) 0.08 15/15 3.24 206 A (R)(K)(YE)(H) A(15) 56 N (FYWH)(R)(TEVA)(MCG) 280 T A(81) 0.09 9/0 3.65 91 V (R)(K)(E)(Y) T(13) 278 A (R)(KY)(E)(H) P(3)GN 275 S S(94)YA 0.11 2/0 4.68 Table 3. List of disruptive mutations for the top 25% of residues in 1civA, PT(3)G that are at the interface with 1civA1. 167 V V(90) 0.14 31/20 2.95 I(5) T(3)A 193 L L(50) 0.18 13/5 3.26 M(50) 49 G G(93) 0.21 27/27 3.04 .(5)VK 52 G G(93) 0.21 22/22 3.26 .(5)V 54 I I(92) 0.21 21/4 2.90 .(4) V(1) L(1)T 150 Q Q(88) 0.22 6/0 3.39 T(5)SY H(1)VNK MI 171 C A(70) 0.23 2/0 4.00 C(20) D(3) S(1) V(3).T

Table 4. The top 25% of residues in 1civA at the interface with NAP.(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: Fig. 5. Residues in 1civA, at the interface with 1civA1, colored by their rela- distance of closest apporach to the ligand. ) tive importance. 1civA1 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1civA.)

4 Table 5. res type disruptive mutations 143 N (TY)(E)(SFVCAWG)(MHDR) 196 L (YR)(TH)(SKECG)(FQWD) 197 D (R)(FWH)(YVCAG)(K) 169 N (Y)(H)(FW)(R) 146 I (YR)(H)(T)(KE) 276 S (KR)(YH)(FEQW)(M) 126 G (R)(K)(E)(H) 225 H (E)(TD)(Q)(M) 168 G (KER)(QHD)(FYMW)(N) 280 T (R)(K)(H)(FW) 275 S (KR)(Q)(H)(M) 167 V (R)(K)(YE)(H) 193 L (Y)(R)(TH)(SCG) 49 G (E)(R)(FKWHD)(Y) 52 G (KER)(HD)(Q)(FMW) 54 I (R)(Y)(H)(K) 150 Q (Y)(H)(FW)(T) 171 C (R)(K)(E)(H)

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

Fig. 6. Residues in 1civA, at the interface with NAP, colored by their relative importance. The ligand (NAP) 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 1civA.)

Figure 6 shows residues in 1civA colored by their importance, at the interface with 1civNAP386.

5 2.4.3 Possible novel functional surfaces at 25% coverage. One Table 6. continued group of residues is conserved on the 1civA surface, away from (or res type substitutions(%) cvg susbtantially larger than) other functional sites and interfaces reco- S(1)T gnizable in PDB entry 1civ. It is shown in Fig. 7. The right panel 275 S S(94)YAPT(3)G 0.11 shows (in blue) the rest of the larger cluster this surface belongs to. 129 P P(98)ASKF 0.12 139 L L(87)V(11)YSIM 0.12 354 E E(94)D(4)AR. 0.12 135 E E(87)ID(4)V(3) 0.13 L(1)NQKH 134 M M(90)Q(4)AE(2) 0.14 T(1)NV 167 V V(90)I(5)T(3)A 0.14 269 I I(91)VL(6).TM 0.14 273 G K(28)G(70)RQ 0.14 232 D ID(97)YSAG 0.15 93 M M(92)A(2)Y.L(2) 0.16 FI 229 Q Q(72)M(26)VHIL 0.16 Fig. 7. A possible active surface on the chain 1civA. The larger cluster it 101 P P(95)GRTE.DLAQ 0.17 belongs to is shown in blue. 193 L L(50)M(50) 0.18 103 L L(92)V(5)IF.C 0.19 195 R R(91)LGQM(3)TW 0.19 The residues belonging to this surface ”patch” are listed in Table K(1)HY 6, while Table 7 suggests possible disruptive replacements for these 268 L I(59)L(13)V(27) 0.20 residues (see Section 3.6). . 302 S S(78)T(18)C(1)A 0.20 Table 6. VP. res type substitutions(%) cvg 49 G G(93).(5)VK 0.21 136 R R(99)KL 0.02 52 G G(93).(5)V 0.21 143 N N(99)H 0.02 54 I I(92).(4)V(1) 0.21 196 L L(99)I 0.02 L(1)T 197 D D(99)E 0.02 261 V V(85)I(13)LM. 0.21 264 R R(99). 0.02 150 Q Q(88)T(5)SYH(1) 0.22 97 D D(99). 0.03 VNKMI 173 T T(99)FS 0.03 56 N Y(80)N(13).(4) 0.23 200 R R(99)K 0.03 S(1) 94 E E(99)Q. 0.04 138 D D(85)A(7)E(4) 0.24 169 N N(99)GDM 0.04 Q(1)GFN 279 S S(99)EC 0.04 231 P GP(84)A(13)VDS 0.24 133 G G(99)CWS 0.05 91 V T(4)V(82)L(7) 0.25 146 I I(98)LV 0.05 I(2)C(2). 90 G G(94)A(5). 0.06 278 A A(69)L(13)I 0.25 199 N N(98)LD 0.06 M(13)G(1)FVS 276 S S(98)ARP 0.06 277 A A(96)ETVS 0.06 126 G G(92)A(5)SDC 0.07 Table 6. Residues forming surface ”patch” in 1civA. 225 H AH(99)PR 0.07 272 W R(85)W(13)S. 0.07 Table 7. 168 G G(84)A(15) 0.08 265 G G(96).A(3)V 0.09 res type disruptive 280 T A(81)T(13)P(3)G 0.09 mutations N 136 R (T)(Y)(D)(S) 130 R R(93)MIL(3)ATK 0.10 143 N (TY)(E)(SFVCAWG)(MHDR) 355 L L(96)AI(2). 0.10 196 L (YR)(TH)(SKECG)(FQWD) 99 L A(83)L(13)C(1). 0.11 197 D (R)(FWH)(YVCAG)(K) 264 R (TD)(SVCLAPIG)(YE)(FMW) continued in next column 97 D (R)(FWH)(VCAG)(KY) continued in next column

6 Table 7. continued 3 NOTES ON USING TRACE RESULTS res type disruptive 3.1 Coverage mutations 173 T (K)(R)(Q)(M) Trace results are commonly expressed in terms of coverage: the resi- 200 R (T)(YD)(SVCAG)(FELWPI) due is important if its “coverage” is small - that is if it belongs to 94 E (FWH)(YVCAG)(TR)(S) some small top percentage of residues [100% is all of the residues 169 N (Y)(H)(FW)(R) in a chain], according to trace. The ET results are presented in the 279 S (R)(K)(FWH)(Q) form of a table, usually limited to top 25% percent of residues (or 133 G (K)(ER)(Q)(MD) to some nearby percentage), sorted by the strength of the presumed 146 I (YR)(H)(T)(KE) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 90 G (KER)(HD)(Q)(FMW) pressure on the residue.) Starting from the top of that list, mutating a 199 N (Y)(H)(FW)(TR) couple of residues should affect the protein somehow, with the exact 276 S (KR)(YH)(FEQW)(M) effects to be determined experimentally. 277 A (R)(K)(Y)(H) 126 G (R)(K)(E)(H) 3.2 Known substitutions 225 H (E)(TD)(Q)(M) One of the table columns is “substitutions” - other amino acid types 272 W (E)(K)(D)(TQ) seen at the same position in the alignment. These amino acid types 168 G (KER)(QHD)(FYMW)(N) may be interchangeable at that position in the protein, so if one wants 265 G (KER)(QHD)(FYMW)(N) to affect the protein by a point mutation, they should be avoided. For 280 T (R)(K)(H)(FW) example if the substitutions are “RVK” and the original protein has 130 R (Y)(T)(D)(E) an R at that position, it is advisable to try anything, but RVK. Conver- 355 L (YR)(H)(T)(KE) sely, when looking for substitutions which will not affect the protein, 99 L (R)(Y)(H)(K) one may try replacing, R with K, or (perhaps more surprisingly), with 275 S (KR)(Q)(H)(M) V. The percentage of times the substitution appears in the alignment 129 P (Y)(R)(T)(H) is given in the immediately following bracket. No percentage is given 139 L (R)(Y)(H)(K) in the cases when it is smaller than 1%. This is meant to be a rough 354 E (FWH)(Y)(CG)(VAR) guide - due to rounding errors these percentages often do not add up 135 E (Y)(H)(FW)(R) to 100%. 134 M (Y)(H)(R)(T) 167 V (R)(K)(YE)(H) 3.3 Surface 269 I (R)(Y)(H)(T) To detect candidates for novel functional interfaces, first we look for 273 G (FEW)(YD)(H)(MR) residues that are solvent accessible (according to DSSP program) by 232 D (R)(H)(K)(FW) 2 at least 10A˚ , which is roughly the area needed for one water mole- 93 M (Y)(T)(HR)(CG) cule to come in the contact with the residue. Furthermore, we require 229 Q (Y)(T)(H)(FW) that these residues form a “cluster” of residues which have neighbor 101 P (Y)(R)(H)(T) within 5A˚ from any of their heavy atoms. 193 L (Y)(R)(TH)(SCG) Note, however, that, if our picture of protein evolution is correct, 103 L (R)(Y)(H)(T) the neighboring residues which are not surface accessible might be 195 R (D)(T)(E)(Y) equally important in maintaining the interaction specificity - they 268 L (YR)(H)(T)(KE) should not be automatically dropped from consideration when choo- 302 S (R)(K)(H)(Q) sing the set for mutagenesis. (Especially if they form a cluster with 49 G (E)(R)(FKWHD)(Y) the surface residues.) 52 G (KER)(HD)(Q)(FMW) 54 I (R)(Y)(H)(K) 3.4 Number of contacts 261 V (Y)(R)(H)(KE) 150 Q (Y)(H)(FW)(T) Another column worth noting is denoted “noc/bb”; it tells the num- 56 N (FYWH)(R)(TEVA)(MCG) ber of contacts heavy atoms of the residue in question make across 138 D (R)(H)(FW)(Y) the interface, as well as how many of them are realized through the 231 P (R)(Y)(H)(K) backbone atoms (if all or most contacts are through the backbone, 91 V (R)(K)(E)(Y) mutation presumably won’t have strong impact). Two heavy atoms 278 A (R)(KY)(E)(H) are considered to be “in contact” if their centers are closer than 5A˚ .

Table 7. Disruptive mutations for the surface patch in 1civA. 3.5 Annotation If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site (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).

7 3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following COVERAGE properties: small [AV GSTC], medium [LPNQDEMIK], large [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- V tively [KHR], or negatively [DE] charged, aromatic [WFYH], 100% 50% 30% 5% long aliphatic chain [EKRQM], OH-group possession [SDETY ], and NH2 group possession [NQRK]. The suggestions are listed according to how different they appear to be from the original amino acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid V types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- RELATIVE IMPORTANCE tive to the fold rather than to the interaction. Many researcher will choose, however, the straightforward alanine mutations, especially in the beginning stages of their investigation. Fig. 8. Coloring scheme used to color residues by their relative importance.

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- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two 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, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant 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 • variability has two subfields: by HHMI/Washington University School of Medicine, 1992-2001, 1. number of different amino acids appearing in in this column and freely distributed under the GNU General Public License. of the alignment 4.3.2 CE To map ligand binding sites from different 2. their type source structures, report maker uses the CE program: • rho ET score - the smaller this value, the lesser variability of http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) this position across the branches of the tree (and, presumably, ”Protein structure alignment by incremental combinatorial extension the greater the importance for the protein) (CE) of the optimal path . Protein Engineering 11(9) 739-747. • cvg coverage - percentage of the residues on the structure which 4.3.3 DSSP In this work a residue is considered solvent accessi- have this rho or smaller ble if the DSSP program finds it exposed to water by at least 10A˚ 2, • gaps percentage of gaps in this column 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. 4.2 Color schemes used Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following color scheme is used in figures with residues colored by [email protected] November 18,2002, by cluster size: black is a single-residue cluster; clusters composed of http://www.cmbi.kun.nl/gv/dssp/descrip.html. more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.4 HSSP Whenever available, report maker uses HSSP ali- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, gnment as a starting point for the analysis (sequences shorter than bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, 75% of the query are taken out, however); R. Schneider, A. de DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, Daruvar, and C. Sander. ”The HSSP database of protein structure- tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 8. http://swift.cmbi.kun.nl/swift/hssp/ 4.3 Credits 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- 4.3.1 Alistat alistat reads a multiple sequence alignment from the Wesley,” Reading, Mass. (1986). file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4.3.6 Muscle When making alignments “from scratch”, report of residues, the average and range of the sequence lengths, and the maker uses Muscle alignment program: Edgar, Robert C. (2004),

8 ”MUSCLE: multiple sequence alignment with high accuracy and report maker itself is described in Mihalek I., I. Res and O. high throughput.” Nucleic Acids Research 32(5), 1792-97. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics http://www.drive5.com/muscle/ 22:1656-7. 4.3.7 Pymol The figures in this report were produced using 4.6 About report maker Pymol. The scripts can be found in the attachment. Pymol report maker was written in 2006 by Ivana Mihalek. The 1D ran- is an open-source application copyrighted by DeLano Scien- king visualization program was written by Ivica Res.ˇ report maker tific LLC (2005). For more information about Pymol see is copyrighted by Lichtarge Lab, Baylor College of Medicine, http://pymol.sourceforge.net/. (Note for Windows Houston. users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) 4.7 Attachments The following files should accompany this report: 4.4 Note about ET Viewer • 1civA.complex.pdb - coordinates of 1civA with all of its inter- Dan Morgan from the Lichtarge lab has developed a visualization acting partners tool specifically for viewing trace results. If you are interested, please • visit: 1civA.etvx - ET viewer input file for 1civA • 1civA.cluster report.summary - Cluster report summary for http://mammoth.bcm.tmc.edu/traceview/ 1civA • The viewer is self-unpacking and self-installing. Input files to be used 1civA.ranks - Ranks file in sequence order for 1civA with ETV (extension .etvx) can be found in the attachment to the • 1civA.clusters - Cluster descriptions for 1civA main report. • 1civA.msf - the multiple sequence alignment used for the chain 1civA 4.5 Citing this work • 1civA.descr - description of sequences used in 1civA msf 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 1civA.ranks sorted - full listing of residues and their ranking for Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 1civA by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1civA.1civA1.if.pml - Pymol script for Figure 5 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1civA.cbcvg - used by other 1civA – related pymol scripts tionary Trace Method Defines Binding Surfaces Common to Protein • 1civA.1civNAP386.if.pml - Pymol script for Figure 6 Families” J. Mol. Bio. 257: 342-358.

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