Pages 1–8 1a59 Evolutionary trace report by report maker August 8, 2010

4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 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 Data Bank entry (PDB id 1a59): Title: Cold-active Compound: Mol id: 1; molecule: citrate synthase; chain: a; engineered: yes Organism, scientific name: Antarctic Bacterium Ds2-3r; 1a59 contains a single unique chain 1a59A (377 residues long).

2 CHAIN 1A59A 2.1 O34002 overview From SwissProt, id O34002, 100% identical to 1a59A: CONTENTS Description: Citrate synthase (EC 2.3.3.1). 1 Introduction 1 Organism, scientific name: Antarctic bacterium DS2-3R. Taxonomy: . 2 Chain 1a59A 1 Catalytic activity: Acetyl-CoA + H(2)O + oxaloacetate = citrate + 2.1 O34002 overview 1 CoA. 2.2 Multiple sequence alignment for 1a59A 1 Biophysicochemical properties: 2.3 Residue ranking in 1a59A 1 Temperature dependence: Optimum temperature is 31 degrees Cel- 2.4 Top ranking residues in 1a59A and their position on sius. Cold-active. Is rapidly inactivated at 45 degrees Celsius, and the structure 2 shows significant activity at 10 degrees Celsius and below; 2.4.1 Clustering of residues at 25% coverage. 2 Pathway: Tricarboxylic acid cycle. 2.4.2 Overlap with known functional surfaces at Subunit: Homodimer. 25% coverage. 2 Miscellaneous: Citrate synthase is found in nearly all cells capable of oxidative metabolism. 3 Notes on using trace results 6 Similarity: Belongs to the citrate synthase family. 3.1 Coverage 6 About: This Swiss-Prot entry is copyright. It is produced through a 3.2 Known substitutions 6 collaboration between the Swiss Institute of Bioinformatics and the 3.3 Surface 6 EMBL outstation - the European Bioinformatics Institute. There are 3.4 Number of contacts 6 no restrictions on its use as long as its content is in no way modified 3.5 Annotation 6 and this statement is not removed. 3.6 Mutation suggestions 6 2.2 Multiple sequence alignment for 1a59A 4 Appendix 7 For the chain 1a59A, the alignment 1a59A.msf (attached) with 120 4.1 File formats 7 sequences was used. The alignment was assembled through combi- 4.2 Color schemes used 7 nation of BLAST searching on the UniProt database and alignment 4.3 Credits 7 using Muscle program. It can be found in the attachment to this 4.3.1 Alistat 7 report, under the name of 1a59A.msf. Its statistics, from the alistat 4.3.2 CE 7 program are the following:

1 Lichtarge lab 2006 by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 2-189 in 1a59A colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 2. Residues 190-378 in 1a59A colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 3. Residues in 1a59A, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 120 Total number of residues: 43727 Smallest: 319 Largest: 377 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Average length: 364.4 top 25% of all residues, this time colored according to clusters they Alignment length: 377 belong to. The clusters in Fig.4 are composed of the residues listed Average identity: 40% in Table 1. Most related pair: 99% Most unrelated pair: 26% Table 1. Most distant seq: 39% cluster size member color residues red 83 8,9,17,19,20,22,23,27,29,31 Furthermore, 3% of residues show as conserved in this alignment. 32,33,47,51,52,55,58,92,93 The alignment consists of 5% eukaryotic ( 3% plantae), 85% 94,180,182,183,185,186,187 prokaryotic, and 10% archaean sequences. (Descriptions of some 189,190,191,192,193,196,200 sequences were not readily available.) The file containing the 201,212,214,216,218,219,220 sequence descriptions can be found in the attachment, under the name 221,222,223,224,225,265,266 1a59A.descr. 267,268,269,270,271,272,276 277,278,279,302,306,315,317 2.3 Residue ranking in 1a59A 318,319,320,321,322,323,324 The 1a59A sequence is shown in Figs. 1–2, with each residue colored 327,337,338,341,342,345,348 according to its estimated importance. The full listing of residues 349,351,352,355,356,362,364 in 1a59A can be found in the file called 1a59A.ranks sorted in the 365 attachment. blue 5 149,158,159,160,162 yellow 2 369,371 2.4 Top ranking residues in 1a59A and their position on the structure Table 1. Clusters of top ranking residues in 1a59A. In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1a59A colored

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) S(3) R(1) 220 L L(59) 0.18 27/16 2.91 S(2) K(14) A(23) 265 M M(76) 0.23 27/10 3.13 I(13) S(5) A(1)FW P(1)

Table 2. The top 25% of residues in 1a59A at the interface with .(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. )

Fig. 4. Residues in 1a59A, colored according to the cluster they belong to: Table 3. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The res type disruptive corresponding Pymol script is attached. mutations 221 H (E)(TQMD)(SNKVCLAPIG)(YR) 224 A (KYER)(QHD)(N)(FTMW) 2.4.2 Overlap with known functional surfaces at 25% coverage. 266 G (KER)(FQMWHD)(NYLPI)(SVA) The name of the ligand is composed of the source PDB identifier 269 H (E)(TQMD)(SNKVCLAPIG)(YR) and the heteroatom name used in that file. 318 N (Y)(FTWH)(SEVCARG)(MD) Coenzyme a . Table 2 lists the top 25% of residues at 320 D (R)(FWH)(YVCAG)(K) the interface with 1a59COA380 (coenzyme a). The following table 268 G (KER)(QHD)(FYMW)(N) (Table 3) suggests possible disruptive replacements for these residues 267 F (KE)(T)(R)(D) (see Section 3.6). 270 R (TYD)(E)(SCG)(FVLAWPI) 315 I (Y)(R)(TH)(CG) Table 2. 219 P (Y)(R)(H)(TE) res type subst’s cvg noc/ dist 220 L (Y)(R)(H)(T) ˚ (%) bb (A) 265 M (Y)(TR)(H)(CG) 221 H H(100) 0.03 9/9 3.79 224 A A(100) 0.03 8/3 3.43 Table 3. List of disruptive mutations for the top 25% of residues in 266 G G(100) 0.03 8/8 2.83 1a59A, that are at the interface with coenzyme a. 269 H H(100) 0.03 19/19 3.54 318 N N(100) 0.03 10/0 3.08 320 D D(95) 0.04 3/0 4.19 Figure 5 shows residues in 1a59A colored by their importance, at the E(4) interface with 1a59COA380. 268 G G(99)V 0.05 24/24 3.25 binding site. Table 4 lists the top 25% of residues at the 267 F F(96)IL 0.08 28/26 2.80 interface with 1a59CIT379 (citric acid). The following table (Table VM 5) suggests possible disruptive replacements for these residues (see 270 R R(80) 0.09 25/8 3.07 Section 3.6). P(13) Table 4. A(6) res type subst’s cvg noc/ dist 315 I I(25) 0.11 18/0 3.51 (%) bb (A˚ ) L(61) 186 H H(99)Q 0.03 34/5 2.75 M(13) 221 H H(100) 0.03 39/11 2.81 219 P AP(94) 0.15 8/8 3.51 222 G G(100) 0.03 9/9 3.78 continued in next column 269 H H(100) 0.03 24/0 2.89 continued in next column

3 Table 5. res type disruptive mutations 186 H (TE)(D)(SVMCAG)(QLPI) 221 H (E)(TQMD)(SNKVCLAPIG)(YR) 222 G (KER)(FQMWHD)(NYLPI)(SVA) 269 H (E)(TQMD)(SNKVCLAPIG)(YR) 278 R (TD)(SYEVCLAPIG)(FMW)(N) 320 D (R)(FWH)(YVCAG)(K) 189 N (Y)(H)(FW)(R) 341 F (KE)(TQD)(SNCG)(R) 345 R (TD)(SVCLAPIG)(YE)(FMW) 190 A (YE)(R)(K)(H) 270 R (TYD)(E)(SCG)(FVLAWPI) 220 L (Y)(R)(H)(T)

Table 5. List of disruptive mutations for the top 25% of residues in 1a59A, that are at the interface with citric acid.

Fig. 5. Residues in 1a59A, at the interface with coenzyme a, colored by their relative importance. The ligand (coenzyme a) 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 1a59A.)

Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) 278 R R(100) 0.03 15/0 2.78 320 D D(95) 0.04 9/0 2.53 E(4) 189 N N(94) 0.05 28/5 3.10 P(4) T(1) 341 F F(99). 0.06 16/0 3.48 345 R R(99). 0.06 12/0 2.83 190 A A(86) 0.09 1/1 4.96 C(11) N(1) 270 R R(80) 0.09 1/0 4.73 Fig. 6. Residues in 1a59A, at the interface with citric acid, colored by their P(13) relative importance. The ligand (citric acid) is colored green. Atoms further A(6) than 30A˚ away from the geometric center of the ligand, as well as on the line 220 L L(59) 0.18 2/2 4.04 of sight to the ligand were removed. (See Appendix for the coloring scheme S(2) for the protein chain 1a59A.) K(14) A(23) Figure 6 shows residues in 1a59A colored by their importance, at the Table 4. The top 25% of residues in 1a59A at the interface with citric interface with 1a59CIT379. acid.(Field names: res: residue number in the PDB entry; type: amino acid Interface with 1a59A1.Table 6 lists the top 25% of residues at type; substs: substitutions seen in the alignment; with the percentage of each the interface with 1a59A1. The following table (Table 7) suggests type in the bracket; noc/bb: number of contacts with the ligand, with the num- possible disruptive replacements for these residues (see Section 3.6). ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

4 Table 6. Table 6. continued res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) 186 H H(99)Q 0.03 7/4 3.34 C(3) 218 G G(100) 0.03 30/30 3.08 T(1) 221 H H(100) 0.03 16/7 3.00 S(1) 269 H H(100) 0.03 1/0 4.36 N(4) 216 L L(98) 0.04 2/2 4.87 220 L L(59) 0.18 46/12 3.13 M(1) S(2) 189 N N(94) 0.05 23/17 3.27 K(14) P(4) A(23) T(1) 369 Y Y(88) 0.18 97/14 3.02 364 R R(99). 0.06 123/33 2.99 .(5) 33 G G(98)ED 0.07 60/60 2.84 F(4) 355 E E(97) 0.07 1/0 4.91 M(1)W D(1). 19 I V(6) 0.20 6/6 4.25 365 P P(98)Q. 0.08 75/30 3.27 I(65) 190 A A(86) 0.09 22/13 3.45 L(27) C(11) 92 H H(85)N 0.20 12/2 3.81 N(1) S(1) 270 R R(80) 0.09 45/1 2.80 D(8)TR P(13) E(1) A(6) 17 T T(40) 0.21 61/48 2.85 200 S S(82) 0.10 5/4 3.51 S(57) G(13) K(1). A(4) 32 R R(86) 0.21 35/31 2.82 371 G G(93) 0.10 16/16 3.51 C(2) .(5) A(5) D(1) V(4) 193 F F(56) 0.13 29/0 3.72 G(1) S(31) 9 L L(59) 0.22 44/14 3.53 T(5) F(29) A(5) Y(8) N(1) .(3) 201 T T(64) 0.13 82/40 2.67 20 S S(34) 0.23 16/16 3.22 S(34)A T(38) 51 L L(93) 0.14 1/0 4.45 C(27) I(3)FMV 271 V V(84) 0.23 12/0 3.53 R L(2) 204 D D(66) 0.14 21/2 2.78 I(9) N(29) E(3) S(2)G 187 S S(10) 0.24 20/6 2.56 8 G G(82) 0.15 40/40 3.16 E(72) A(14) G(10) .(3) T(5)D 219 P AP(94) 0.15 24/8 3.44 356 Q Q(55) 0.24 1/0 4.96 S(3) M(27) R(1) Y(7) 362 L L(24) 0.15 29/16 2.80 F(2) I(75). H(2) 214 G G(55) 0.16 22/22 3.04 S(3).E A(33) 55 S G(88) 0.25 5/5 3.34 continued in next column Y(2) R(1) L(2) continued in next column

5 Table 6. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) D(1)S E(1)Q

Table 6. The top 25% of residues in 1a59A at the interface with 1a59A1. (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 7. res type disruptive mutations 186 H (TE)(D)(SVMCAG)(QLPI) 218 G (KER)(FQMWHD)(NYLPI)(SVA) 221 H (E)(TQMD)(SNKVCLAPIG)(YR) 269 H (E)(TQMD)(SNKVCLAPIG)(YR) 216 L (Y)(R)(TH)(SCG) Fig. 7. Residues in 1a59A, at the interface with 1a59A1, colored by their rela- 189 N (Y)(H)(FW)(R) tive importance. 1a59A1 is shown in backbone representation (See Appendix 364 R (TD)(SVCLAPIG)(YE)(FMW) for the coloring scheme for the protein chain 1a59A.) 33 G (R)(FKWH)(Y)(Q) 355 E (FW)(H)(VCAG)(R) 365 P (Y)(R)(TH)(CG) 3 NOTES ON USING TRACE RESULTS 190 A (YE)(R)(K)(H) 3.1 Coverage 270 R (TYD)(E)(SCG)(FVLAWPI) 200 S (KR)(QH)(FMW)(E) Trace results are commonly expressed in terms of coverage: the resi- 371 G (R)(K)(FWH)(M) due is important if its “coverage” is small - that is if it belongs to 193 F (K)(E)(Q)(R) some small top percentage of residues [100% is all of the residues 201 T (KR)(QH)(FMW)(E) in a chain], according to trace. The ET results are presented in the 51 L (Y)(R)(T)(H) form of a table, usually limited to top 25% percent of residues (or 204 D (R)(FWH)(KY)(VMA) to some nearby percentage), sorted by the strength of the presumed 8 G (KER)(HD)(Q)(FMW) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 219 P (Y)(R)(H)(TE) pressure on the residue.) Starting from the top of that list, mutating a 362 L (Y)(R)(T)(H) couple of residues should affect the protein somehow, with the exact 214 G (R)(K)(E)(H) effects to be determined experimentally. 220 L (Y)(R)(H)(T) 369 Y (K)(Q)(E)(NR) 3.2 Known substitutions 19 I (YR)(H)(T)(KE) One of the table columns is “substitutions” - other amino acid types 92 H (E)(M)(T)(Q) seen at the same position in the alignment. These amino acid types 17 T (FWR)(KH)(M)(Q) may be interchangeable at that position in the protein, so if one wants 32 R (D)(E)(Y)(T) to affect the protein by a point mutation, they should be avoided. For 9 L (R)(TK)(YE)(SCHG) example if the substitutions are “RVK” and the original protein has 20 S (KR)(FQMWH)(E)(NLPI) an R at that position, it is advisable to try anything, but RVK. Conver- 271 V (YR)(KH)(E)(Q) sely, when looking for substitutions which will not affect the protein, 187 S (R)(K)(FWH)(M) one may try replacing, R with K, or (perhaps more surprisingly), with 356 Q (Y)(T)(H)(FW) V. The percentage of times the substitution appears in the alignment 55 S (R)(K)(FWH)(M) is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough Table 7. List of disruptive mutations for the top 25% of residues in guide - due to rounding errors these percentages often do not add up 1a59A, that are at the interface with 1a59A1. to 100%. 3.3 Surface Figure 7 shows residues in 1a59A colored by their importance, at the To detect candidates for novel functional interfaces, first we look for interface with 1a59A1. residues that are solvent accessible (according to DSSP program) by

6 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, COVERAGE the neighboring residues which are not surface accessible might be

equally important in maintaining the interaction specificity - they V should not be automatically dropped from consideration when choo- 100% 50% 30% 5% sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across V the interface, as well as how many of them are realized through the RELATIVE IMPORTANCE backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . Fig. 8. Coloring scheme used to color residues by their relative importance. 3.5 Annotation If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller • gaps Mutation suggestions are completely heuristic and based on comple- percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 8. disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will 4.3 Credits choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4 APPENDIX of residues, the average and range of the sequence lengths, and the 4.1 File formats alignment length (e.g. including gap characters). Also shown are Files with extension “ranks sorted” are the actual trace results. The some percent identities. A percent pairwise alignment identity is defi- fields in the table in this file: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two • alignment# number of the position in the alignment sequences. The ”average percent identity”, ”most related pair”, and • residue# residue number in the PDB file ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • type amino acid type seq” is calculated by finding the maximum pairwise identity (best • rank rank of the position according to older version of ET relative) for all N sequences, then finding the minimum of these N • variability has two subfields: numbers (hence, the most outlying sequence). alistat is copyrighted 1. number of different amino acids appearing in in this column by HHMI/Washington University School of Medicine, 1992-2001, of the alignment and freely distributed under the GNU General Public License.

7 4.3.2 CE To map ligand binding sites from different The viewer is self-unpacking and self-installing. Input files to be used source structures, report maker uses the CE program: with ETV (extension .etvx) can be found in the attachment to the http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) main report. ”Protein structure alignment by incremental combinatorial extension 4.5 Citing this work (CE) of the optimal path . Protein Engineering 11(9) 739-747. The method used to rank residues and make predictions in this report 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ble if the DSSP program finds it exposed to water by at least 10A˚ , Evolution-Entropy Hybrid Methods for Ranking of Protein Residues which is roughly the area needed for one water molecule to come in by Importance” J. Mol. Bio. 336: 1265-82. For the original version the contact with the residue. DSSP is copyrighted by W. Kabsch, C. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version tionary Trace Method Defines Binding Surfaces Common to Protein by [email protected] November 18,2002, Families” J. Mol. Bio. 257: 342-358. http://www.cmbi.kun.nl/gv/dssp/descrip.html. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- of service for comparative analysis of .” Bioinformatics gnment as a starting point for the analysis (sequences shorter than 22:1656-7. 75% of the query are taken out, however); R. Schneider, A. de 4.6 About report maker Daruvar, and C. Sander. ”The HSSP database of protein structure- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker http://swift.cmbi.kun.nl/swift/hssp/ is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- 4.7 Attachments Wesley,” Reading, Mass. (1986). The following files should accompany this report: 4.3.6 Muscle When making alignments “from scratch”, report • 1a59A.complex.pdb - coordinates of 1a59A with all of its maker uses Muscle alignment program: Edgar, Robert C. (2004), interacting partners ”MUSCLE: multiple sequence alignment with high accuracy and • 1a59A.etvx - ET viewer input file for 1a59A high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1a59A.cluster report.summary - Cluster report summary for http://www.drive5.com/muscle/ 1a59A • 4.3.7 Pymol The figures in this report were produced using 1a59A.ranks - Ranks file in sequence order for 1a59A Pymol. The scripts can be found in the attachment. Pymol • 1a59A.clusters - Cluster descriptions for 1a59A is an open-source application copyrighted by DeLano Scien- • 1a59A.msf - the multiple sequence alignment used for the chain tific LLC (2005). For more information about Pymol see 1a59A http://pymol.sourceforge.net/. (Note for Windows • 1a59A.descr - description of sequences used in 1a59A msf users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) • 1a59A.ranks sorted - full listing of residues and their ranking for 1a59A 4.4 Note about ET Viewer • 1a59A.1a59COA380.if.pml - Pymol script for Figure 5 Dan Morgan from the Lichtarge lab has developed a visualization • 1a59A.cbcvg - used by other 1a59A – related pymol scripts tool specifically for viewing trace results. If you are interested, please • visit: 1a59A.1a59CIT379.if.pml - Pymol script for Figure 6 • 1a59A.1a59A1.if.pml - Pymol script for Figure 7 http://mammoth.bcm.tmc.edu/traceview/

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