Pages 1–8 2qtl Evolutionary trace report by report maker June 23, 2010

4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 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 2qtl): Title: Crystal structure of the fad-containing fnr-like module of human methionine synthase reductase Compound: Mol id: 1; molecule: methionine synthase reductase; CONTENTS chain: a; fragment: fnr-like module; synonym: methionine synt- hase reductase, mitochondrial; msr; ec: 1.16.1.8; engineered: yes; 1 Introduction 1 mutation: yes Organism, scientific name: Homo Sapiens; 2 Chain 2qtlA 1 2qtl contains a single unique chain 2qtlA (448 residues long). 2.1 Q7Z4M8 overview 1 2.2 Multiple sequence alignment for 2qtlA 1 2.3 Residue ranking in 2qtlA 1 2.4 Top ranking residues in 2qtlA and their position on the structure 1 2 CHAIN 2QTLA 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at 2.1 Q7Z4M8 overview 25% coverage. 2 From SwissProt, id Q7Z4M8, 84% identical to 2qtlA: 2.4.3 Possible novel functional surfaces at 25% Description: MTRR protein (Fragment). coverage. 4 Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3 Notes on using trace results 6 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 3.1 Coverage 6 Catarrhini; Hominidae; Homo. 3.2 Known substitutions 6 3.3 Surface 6 3.4 Number of contacts 7 3.5 Annotation 7 2.2 Multiple sequence alignment for 2qtlA 3.6 Mutation suggestions 7 For the chain 2qtlA, the alignment 2qtlA.msf (attached) with 111 sequences was used. The alignment was assembled through combi- 4 Appendix 7 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 7 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 7 report, under the name of 2qtlA.msf. Its statistics, from the alistat 4.3 Credits 7 program are the following:

1 Lichtarge lab 2006 2.4 Top ranking residues in 2qtlA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 2qtlA colored by their 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 217-454 in 2qtlA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 455-698 in 2qtlA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Fig. 3. Residues in 2qtlA, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 111 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 46026 top 25% of all residues, this time colored according to clusters they Smallest: 369 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 448 in Table 1. Average length: 414.6 Table 1. Alignment length: 448 Average identity: 34% cluster size member Most related pair: 99% color residues Most unrelated pair: 17% red 97 283,284,285,291,293,294,295 Most distant seq: 38% 297,307,309,310,311,312,314 319,324,370,375,426,428,448 450,451,452,453,454,455,456 Furthermore, 1% of residues show as conserved in this alignment. 457,458,467,469,470,471,473 The alignment consists of 66% eukaryotic ( 18% vertebrata, 485,487,488,489,490,525,530 6% arthropoda, 18% fungi, 13% plantae), and 32% prokaryotic 532,533,541,542,544,545,546 sequences. (Descriptions of some sequences were not readily availa- 547,548,550,551,552,553,554 ble.) The file containing the sequence descriptions can be found in 555,558,559,572,576,577,578 the attachment, under the name 2qtlA.descr. 579,580,581,586,588,589,592 593,608,609,610,611,622,624 2.3 Residue ranking in 2qtlA 625,626,627,648,649,650,651 652,653,656,657,659,660,664 The 2qtlA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 2qtlA can be found in the file called 2qtlA.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) 652 D D(86) 0.06 1/0 4.79 P(9) G(1)SN 697 W Y(33) 0.06 126/20 3.34 W(48) F(18) 696 I V(79) 0.07 3/3 4.38 I(18) T(1) 547 T T(96) 0.08 3/0 4.47 S(2)A 490 T S(71) 0.09 22/7 2.64 T(28) 375 E E(42) 0.11 1/0 4.97 D(54) S(1)N 312 A A(31) 0.12 3/0 4.14 H(57) C(4) Fig. 4. Residues in 2qtlA, colored according to the cluster they belong to: T(1) red, followed by blue and yellow are the largest clusters (see Appendix for S(3)V the coloring scheme). Clockwise: front, back, top and bottom views. The 473 V V(81) 0.13 18/1 3.71 corresponding Pymol script is attached. L(8) E(7)A M(1) Table 1. continued 525 R E(10) 0.13 4/0 3.44 cluster size member R(62) color residues K(17) 667,691,692,695,696,697 Q(7) blue 9 380,382,385,388,392,401,404 H(2) 413,433 452 P L(18) 0.16 58/34 2.61 P(7) Table 1. Clusters of top ranking residues in 2qtlA. F(9) Y(47) E(12) 2.4.2 Overlap with known functional surfaces at 25% coverage. H(1)MAS The name of the ligand is composed of the source PDB identifier 469 V T(73) 0.16 11/11 2.85 and the heteroatom name used in that file. V(2) FAD binding site. Table 2 lists the top 25% of residues at the inter- I(4) face with 2qtlAFAD700 (fad). The following table (Table 3) suggests C(6) possible disruptive replacements for these residues (see Section 3.6). L(9) Table 2. M(1)AS res type subst’s cvg noc/ dist 488 V G(9) 0.17 24/17 2.88 (%) bb (A˚ ) V(70)T 451 R R(100) 0.02 42/3 2.91 I(2) 454 S S(100) 0.02 23/12 2.81 H(3) 487 G G(100) 0.02 21/21 3.26 L(6) 695 D D(88) 0.02 6/0 3.85 Q(6) E(11) 489 C A(38) 0.20 16/12 2.76 550 A A(98) 0.04 2/0 4.74 C(54)N S(1) T(4)V 453 Y Y(97) 0.05 81/24 2.77 470 F V(47) 0.23 13/8 3.21 F(2) continued in next column continued in next column

3 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) F(3) C(13) A(13) S(1) I(15) Y(3)L 471 N G(30) 0.23 8/5 3.59 N(3) A(31) V(26) S(8)

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

Fig. 5. Residues in 2qtlA, at the interface with FAD, colored by their relative Table 3. importance. The ligand (FAD) is colored green. Atoms further than 30A˚ away res type disruptive from the geometric center of the ligand, as well as on the line of sight to the mutations ligand were removed. (See Appendix for the coloring scheme for the protein 451 R (TD)(SYEVCLAPIG)(FMW)(N) chain 2qtlA.) 454 S (KR)(FQMWH)(NYELPI)(D) 487 G (KER)(FQMWHD)(NYLPI)(SVA) 695 D (R)(FWH)(YVCAG)(K) 550 A (KR)(YE)(QH)(D) 453 Y (K)(Q)(EM)(NR) 652 D (R)(H)(FW)(Y) 697 W (K)(E)(Q)(D) 696 I (R)(Y)(H)(K) 547 T (KR)(QH)(FMW)(E) 490 T (KR)(FQMWH)(NELPI)(D) 375 E (FWH)(R)(Y)(VCAG) 312 A (K)(ER)(YQ)(D) 473 V (Y)(R)(KH)(E) Fig. 6. A possible active surface on the chain 2qtlA. The larger cluster it 525 R (T)(Y)(VCADG)(S) belongs to is shown in blue. 452 P (R)(Y)(T)(KH) 469 V (R)(KY)(E)(H) 488 V (R)(YE)(K)(H) The residues belonging to this surface ”patch” are listed in Table 489 C (R)(KE)(H)(Q) 4, while Table 5 suggests possible disruptive replacements for these 470 F (K)(E)(QR)(D) residues (see Section 3.6). 471 N (Y)(H)(R)(FEW) Table 4. res type substitutions(%) cvg Table 3. List of disruptive mutations for the top 25% of residues in 2qtlA, that are at the interface with FAD. 451 R R(100) 0.02 454 S S(100) 0.02 487 G G(100) 0.02 Figure 5 shows residues in 2qtlA colored by their importance, at the 546 G G(100) 0.02 interface with 2qtlAFAD700. 579 G G(99)R 0.02 610 S S(100) 0.02 2.4.3 Possible novel functional surfaces at 25% coverage. One 695 D D(88)E(11) 0.02 group of residues is conserved on the 2qtlA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- continued in next column gnizable in PDB entry 2qtl. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

4 Table 4. continued Table 4. continued res type substitutions(%) cvg res type substitutions(%) cvg 530 F F(88)M(9)L(1) 0.03 L(1)FQ 626 Q Q(99)T 0.03 452 P L(18)P(7)F(9) 0.16 319 N N(98)HQ 0.04 Y(47)E(12)H(1)M 611 R R(98)E(1) 0.04 AS 656 M M(88)P(9)I(1) 0.04 469 V T(73)V(2)I(4) 0.16 311 D D(88)E(11) 0.05 C(6)L(9)M(1)AS 453 Y Y(97)F(2) 0.05 659 D D(80)E(7)G(3)N 0.16 533 P P(96)T(1)S(1) 0.05 S(4)H(1)A(1) 625 V V(89)I(10) 0.05 285 T T(27)G(1)H(16) 0.17 622 A K(91)A(3)RP(1) 0.06 N(18)F(8)Q(20) .(1) K(2)L(1)V(1)S 624 Y Y(96)H(1)R(1) 0.06 488 V G(9)V(70)TI(2) 0.17 652 D D(86)P(9)G(1)SN 0.06 H(3)L(6)Q(6) 697 W Y(33)W(48)F(18) 0.06 572 G G(87)TC(3)A(2) 0.17 307 Y Y(93)F(5)I 0.07 P(1).EVN 657 A A(81)E(1)V(9) 0.07 293 T V(14)T(44)C(22) 0.18 S(4)P(1) L(5)I(8)K(4)S 696 I V(79)I(18)T(1) 0.07 392 T .(21)T(19)C(7) 0.18 401 L L(94)A(1)M(2)T 0.08 A(47)S(2)Q 547 T T(96)S(2)A 0.08 448 L L(83)I(8)M(6)TA 0.18 692 Y Y(94)F(3)HL 0.08 653 A M(1)A(51)C(1) 0.18 291 K K(28)R(67)E(1)L 0.09 E(7)G(11)V(12) Q T(9)K(2)S 490 T S(71)T(28) 0.09 428 L L(69)M(11)F(4) 0.19 539 P P(96)QN(1)S 0.09 V(9)I(5) 586 D D(87)E(9).AG 0.09 588 L L(66)I(27)FV 0.19 545 P P(82)L(9)V(2)A 0.10 M(1)HY(1) S(2)N 309 P A(8)P(44)T(21) 0.20 581 R R(78)Q(12)P(6) 0.10 E(7)S(6)I(10) K(1)Y V(1) 589 F Y(90)F(6)K(1)L 0.10 489 C A(38)C(54)NT(4) 0.20 375 E E(42)D(54)S(1)N 0.11 V 312 A A(31)H(57)C(4) 0.12 627 D H(53)D(40)T(1) 0.20 T(1)S(3)V Q(2)NA 532 L L(85)M(5)F(6) 0.12 467 H H(74)T(2)S(14)A 0.21 Y(1)P E(1)R(3)GQ 284 L I(9)L(82)V(8) 0.13 485 R R(39)H(31)N(6) 0.21 473 V V(81)L(8)E(7)A 0.13 V(3)Y(8)F(2) M(1) .(2)K(1)I(1)QL 525 R E(10)R(62)K(17) 0.13 297 E E(79)D(5)GR(7)K 0.22 Q(7)H(2) V(1)Q(1)T(1)S 691 R R(88)K(5)VQ(4)M 0.13 314 S G(69)S(3)A(17) 0.22 450 P P(75)A(10)T(1) 0.14 V(4)NCY(1)ET R(8)Q(2)K 592 E E(81)L(5)Q(2) 0.22 580 C D(14)C(62)S(10) 0.14 F(1)D(4)K(1)YI N(7)A(2)E(1)L 609 F F(72)L(5)H(1) 0.22 295 L H(71)L(16)F(5) 0.15 Y(10)W(7)CE(1) K(1)V(1)AM(1)Q 388 L F(10)L(62)A(9) 0.23 426 D D(54)E(30)H(2) 0.15 M(6)Y(9)I(1) A(6)G(1)Q(1)V 433 S .(14)S(53)T(5) 0.23 R(1) A(9)N(5)P(5)D 559 R R(85)K(9)W(1) 0.15 G(2)IEK(1) continued in next column continued in next column

5 Table 4. continued Table 5. continued res type substitutions(%) cvg res type disruptive 470 F V(47)F(3)C(13) 0.23 mutations A(13)S(1)I(15) 589 F (E)(K)(T)(D) Y(3)L 375 E (FWH)(R)(Y)(VCAG) 471 N G(30)N(3)A(31) 0.23 312 A (K)(ER)(YQ)(D) V(26)S(8) 532 L (R)(Y)(T)(K) 283 Q K(16)Q(6)N(22) 0.25 284 L (YR)(H)(T)(KE) E(34)L(2)R(11) 473 V (Y)(R)(KH)(E) H(1)V(1)F(1)C 525 R (T)(Y)(VCADG)(S) 294 L R(40)L(9)V(1) 0.25 691 R (TY)(D)(CG)(S) M(7)I(23)T(4) 450 P (Y)(HR)(T)(E) F(9)QYC 580 C (R)(K)(H)(FW) 558 H E(59)H(9)A(7) 0.25 295 L (Y)(R)(T)(H) R(2)Q(18)Y(1)F 426 D (R)(FWH)(Y)(K) 559 R (T)(D)(Y)(SECG) Table 4. Residues forming surface ”patch” in 2qtlA. 452 P (R)(Y)(T)(KH) 469 V (R)(KY)(E)(H) 659 D (R)(FWH)(KY)(VCAG) Table 5. 285 T (R)(K)(H)(FW) res type disruptive 488 V (R)(YE)(K)(H) mutations 572 G (R)(K)(E)(H) 451 R (TD)(SYEVCLAPIG)(FMW)(N) 293 T (R)(K)(H)(FW) 454 S (KR)(FQMWH)(NYELPI)(D) 392 T (R)(K)(H)(FW) 487 G (KER)(FQMWHD)(NYLPI)(SVA) 448 L (R)(Y)(H)(TK) 546 G (KER)(FQMWHD)(NYLPI)(SVA) 653 A (R)(Y)(K)(H) 579 G (E)(D)(FKMW)(YQLPHIR) 428 L (R)(Y)(T)(H) 610 S (KR)(FQMWH)(NYELPI)(D) 588 L (R)(Y)(T)(K) 695 D (R)(FWH)(YVCAG)(K) 309 P (R)(Y)(H)(K) 530 F (T)(KE)(DR)(QCG) 489 C (R)(KE)(H)(Q) 626 Q (FYWH)(TVA)(SCDRG)(ELPI) 627 D (R)(FWH)(Y)(K) 319 N (Y)(T)(FW)(SEVCAHG) 467 H (E)(MD)(Q)(TK) 611 R (T)(YVCAG)(SFLWPDI)(EM) 485 R (T)(D)(YE)(SCG) 656 M (Y)(TH)(R)(CG) 297 E (FW)(H)(Y)(R) 311 D (R)(FWH)(YVCAG)(K) 314 S (R)(K)(H)(FQW) 453 Y (K)(Q)(EM)(NR) 592 E (H)(FW)(YR)(CG) 533 P (R)(Y)(H)(K) 609 F (K)(E)(Q)(T) 625 V (YR)(KE)(H)(QD) 388 L (R)(Y)(T)(KH) 622 A (Y)(E)(D)(HR) 433 S (R)(KH)(FW)(Y) 624 Y (M)(K)(Q)(E) 470 F (K)(E)(QR)(D) 652 D (R)(H)(FW)(Y) 471 N (Y)(H)(R)(FEW) 697 W (K)(E)(Q)(D) 283 Q (Y)(T)(H)(FW) 307 Y (K)(Q)(R)(E) 294 L (R)(Y)(H)(T) 657 A (R)(Y)(K)(H) 558 H (E)(T)(D)(M) 696 I (R)(Y)(H)(K) 401 L (R)(Y)(H)(K) Table 5. Disruptive mutations for the surface patch in 2qtlA. 547 T (KR)(QH)(FMW)(E) 692 Y (K)(Q)(E)(M) 291 K (Y)(T)(FW)(CG) 490 T (KR)(FQMWH)(NELPI)(D) 3 NOTES ON USING TRACE RESULTS 539 P (Y)(R)(H)(T) 586 D (R)(H)(FW)(Y) 3.1 Coverage 545 P (YR)(H)(TKE)(CG) Trace results are commonly expressed in terms of coverage: the resi- 581 R (T)(D)(Y)(S) due is important if its “coverage” is small - that is if it belongs to continued in next column some small top percentage of residues [100% is all of the residues in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the

6 pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3.2 Known substitutions COVERAGE One of the table columns is “substitutions” - other amino acid types

seen at the same position in the alignment. These amino acid types V may be interchangeable at that position in the protein, so if one wants 100% 50% 30% 5% to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with V. The percentage of times the substitution appears in the alignment V is given in the immediately following bracket. No percentage is given RELATIVE IMPORTANCE 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%. Fig. 7. Coloring scheme used to color residues by their relative importance. 3.3 Surface To detect candidates for novel functional interfaces, first we look for according to how different they appear to be from the original amino residues that are solvent accessible (according to DSSP program) by acid, and they are grouped in round brackets if they appear equally 2 at least 10A˚ , which is roughly the area needed for one water mole- disruptive. From left to right, each bracketed group of amino acid cule to come in the contact with the residue. Furthermore, we require types resembles more strongly the original (i.e. is, presumably, less that these residues form a “cluster” of residues which have neighbor disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 4.1 File formats Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • ber of contacts heavy atoms of the residue in question make across alignment# number of the position in the alignment the interface, as well as how many of them are realized through the • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 3.5 Annotation 1. number of different amino acids appearing in in this column If the residue annotation is available (either from the pdb file or of the alignment 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 Mutation suggestions are completely heuristic and based on comple- • gaps 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,

7 DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, is an open-source application copyrighted by DeLano Scien- tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. tific LLC (2005). For more information about Pymol see The colors used to distinguish the residues by the estimated http://pymol.sourceforge.net/. (Note for Windows evolutionary pressure they experience can be seen in Fig. 7. users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) 4.3 Credits 4.3.1 Alistat alistat reads a multiple sequence alignment from the 4.4 Note about ET Viewer file and shows a number of simple statistics about it. These stati- Dan Morgan from the Lichtarge lab has developed a visualization stics include the format, the number of sequences, the total number tool specifically for viewing trace results. If you are interested, please of residues, the average and range of the sequence lengths, and the visit: alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- http://mammoth.bcm.tmc.edu/traceview/ ned as (idents / MIN(len1, len2)) where idents is the number of The viewer is self-unpacking and self-installing. Input files to be used exact identities and len1, len2 are the unaligned lengths of the two with ETV (extension .etvx) can be found in the attachment to the sequences. The ”average percent identity”, ”most related pair”, and main report. ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant 4.5 Citing this work seq” is calculated by finding the maximum pairwise identity (best The method used to rank residues and make predictions in this report relative) for all N sequences, then finding the minimum of these N can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of numbers (hence, the most outlying sequence). alistat is copyrighted Evolution-Entropy Hybrid Methods for Ranking of Protein Residues by HHMI/Washington University School of Medicine, 1992-2001, by Importance” J. Mol. Bio. 336: 1265-82. For the original version and freely distributed under the GNU General Public License. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 4.3.2 CE To map ligand binding sites from different tionary Trace Method Defines Binding Surfaces Common to Protein source structures, report maker uses the CE program: Families” J. Mol. Bio. 257: 342-358. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) report maker itself is described in Mihalek I., I. Res and O. ”Protein structure alignment by incremental combinatorial extension Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type (CE) of the optimal path . Protein Engineering 11(9) 739-747. of service for comparative analysis of proteins.” Bioinformatics 22:1656-7. 4.3.3 DSSP In this work a residue is considered solvent accessi- ble if the DSSP program finds it exposed to water by at least 10A˚ 2, 4.6 About report maker which is roughly the area needed for one water molecule to come in report maker was written in 2006 by Ivana Mihalek. The 1D ran- the contact with the residue. DSSP is copyrighted by W. Kabsch, C. king visualization program was written by Ivica Res.ˇ report maker Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version is copyrighted by Lichtarge Lab, Baylor College of Medicine, by [email protected] November 18,2002, Houston. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.7 Attachments 4.3.4 HSSP Whenever available, report maker uses HSSP ali- The following files should accompany this report: gnment as a starting point for the analysis (sequences shorter than • 2qtlA.complex.pdb - coordinates of 2qtlA with all of its interac- 75% of the query are taken out, however); R. Schneider, A. de ting partners Daruvar, and C. Sander. ”The HSSP database of protein structure- • sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 2qtlA.etvx - ET viewer input file for 2qtlA • 2qtlA.cluster report.summary - Cluster report summary for http://swift.cmbi.kun.nl/swift/hssp/ 2qtlA 4.3.5 LaTex The text for this report was processed using LATEX; • 2qtlA.ranks - Ranks file in sequence order for 2qtlA Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 2qtlA.clusters - Cluster descriptions for 2qtlA Wesley,” Reading, Mass. (1986). • 2qtlA.msf - the multiple sequence alignment used for the chain 4.3.6 Muscle When making alignments “from scratch”, report 2qtlA maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2qtlA.descr - description of sequences used in 2qtlA msf ”MUSCLE: multiple sequence alignment with high accuracy and • high throughput.” Nucleic Acids Research 32(5), 1792-97. 2qtlA.ranks sorted - full listing of residues and their ranking for 2qtlA http://www.drive5.com/muscle/ • 2qtlA.2qtlAFAD700.if.pml - Pymol script for Figure 5 4.3.7 Pymol The figures in this report were produced using • 2qtlA.cbcvg - used by other 2qtlA – related pymol scripts Pymol. The scripts can be found in the attachment. Pymol

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