Pages 1–8 1ezw Evolutionary trace report by report maker March 17, 2010

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

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1ezw): Title: Structure of coenzyme f420 dependent tetrahydromethanopte- rin reductase from kandleri Compound: Mol id: 1; molecule: coenzyme f420-dependent n5,n10- methylenetetrahydromethanopterin reductase; chain: a Organism, scientific name: Methanopyrus Kandleri; 1ezw contains a single unique chain 1ezwA (347 residues long).

CONTENTS 2 CHAIN 1EZWA 2.1 Q8TXY4 overview 1 Introduction 1 From SwissProt, id Q8TXY4, 99% identical to 1ezwA: 2 Chain 1ezwA 1 Description: 5,10-methylenetetrahydromethanopterin reductase 2.1 Q8TXY4 overview 1 (EC 1.5.99.11) (Coenzyme F420-dependent N(5),N(10)- 2.2 Multiple sequence alignment for 1ezwA 1 methylenetetrahydromethanopterin reductase) (Methylene-H(4)MPT 2.3 Residue ranking in 1ezwA 1 reductase). 2.4 Top ranking residues in 1ezwA and their position on Organism, scientific name: Methanopyrus kandleri. the structure 1 : ; ; ; ; 2.4.1 Clustering of residues at 25% coverage. 2 ; Methanopyrus. 2.4.2 Overlap with known functional surfaces at Function: Catalyzes the reversible reduction of methylene-H(4)MPT 25% coverage. 2 to methyl-H(4)MPT. 2.4.3 Possible novel functional surfaces at 25% Catalytic activity: 5-methyltetrahydromethanopterin + coenzyme coverage. 5 F420 = 5,10-methylenetetrahydromethanopterin + reduced coen- zyme F420. 3 Notes on using trace results 6 Enzyme regulation: Requires the presence of relatively high con- 3.1 Coverage 6 centrations of either sulfate or phosphate for maximal activity. 3.2 Known substitutions 7 Pathway: Methanogenesis from carbon dioxide; fifth step. 3.3 Surface 7 Subunit: Homotetramer composed of two loosely associated dimers 3.4 Number of contacts 7 (Probable). 3.5 Annotation 7 Subcellular location: Cytoplasmic. 3.6 Mutation suggestions 7 Similarity: Belongs to the mer family. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 7 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 7 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 7 no restrictions on its use as long as its content is in no way modified 4.3 Credits 8 and this statement is not removed.

1 Lichtarge lab 2006 in 1ezwA can be found in the file called 1ezwA.ranks sorted in the attachment. 2.4 Top ranking residues in 1ezwA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1ezwA colored 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 2-174 in 1ezwA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 2. Residues 175-348 in 1ezwA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

2.2 Multiple sequence alignment for 1ezwA For the chain 1ezwA, the alignment 1ezwA.msf (attached) with 33 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1ezwA.msf. Its statistics, from the Fig. 3. Residues in 1ezwA, colored by their relative importance. Clockwise: alistat program are the following: front, back, top and bottom views.

Format: MSF Number of sequences: 33 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 10589 top 25% of all residues, this time colored according to clusters they Smallest: 288 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 347 in Table 1. Average length: 320.9 Alignment length: 347 Table 1. Average identity: 45% cluster size member Most related pair: 98% color residues Most unrelated pair: 19% red 82 7,8,9,10,12,26,27,30,35,39 Most distant seq: 31% 40,42,49,53,56,62,64,65,67 69,85,87,89,90,91,94,96,97 98,99,127,172,174,176,177 Furthermore, 3% of residues show as conserved in this alignment. 178,179,180,182,186,187,191 The alignment consists of 9% prokaryotic, and 30% archaean 192,194,196,197,198,200,202 sequences. (Descriptions of some sequences were not readily availa- 206,210,213,228,229,233,241 ble.) The file containing the sequence descriptions can be found in 244,245,248,249,250,251,252 the attachment, under the name 1ezwA.descr. 255,257,263,264,281,283,290 continued in next column 2.3 Residue ranking in 1ezwA The 1ezwA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues

2 the number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations 56 T (KR)(FQMWH)(NELPI)(D) 90 R (TD)(SYEVCLAPIG)(FMW)(N) 53 A (YR)(K)(H)(E)

Table 3. List of disruptive mutations for the top 25% of residues in 1ezwA, that are at the interface with magnesium ion.

Fig. 4. Residues in 1ezwA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached.

Table 1. continued cluster size member color residues 298,302,304,314,318,319,325 326,327,329,336,338 blue 3 73,77,78

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

Fig. 5. Residues in 1ezwA, at the interface with magnesium ion, colored 2.4.2 Overlap with known functional surfaces at 25% coverage. by their relative importance. The ligand (magnesium ion) is colored green. The name of the ligand is composed of the source PDB identifier Atoms further than 30A˚ away from the geometric center of the ligand, as well and the heteroatom name used in that file. as on the line of sight to the ligand were removed. (See Appendix for the Magnesium ion binding site. Table 2 lists the top 25% of residues coloring scheme for the protein chain 1ezwA.) at the interface with 1ezwAMG361 (magnesium ion). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Figure 5 shows residues in 1ezwA colored by their importance, at the interface with 1ezwAMG361. Table 2. Interface with 1ezwA1.Table 4 lists the top 25% of residues at res type subst’s cvg noc/ dist the interface with 1ezwA1. The following table (Table 5) suggests (%) bb (A˚ ) possible disruptive replacements for these residues (see Section 3.6). 56 T T(100) 0.04 4/4 2.27 Table 4. 90 R R(100) 0.04 2/0 3.95 res type subst’s cvg noc/ dist 53 A A(93) 0.10 2/2 3.58 (%) bb (A˚ ) M(3) 27 E E(100) 0.04 2/0 4.08 S(3) 56 T T(100) 0.04 4/4 3.67 89 G G(100) 0.04 2/2 4.87 Table 2. The top 25% of residues in 1ezwA at the interface with magne- 90 R R(100) 0.04 9/0 3.00 sium ion.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of continued in next column each type in the bracket; noc/bb: number of contacts with the ligand, with

3 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) 53 A A(93) 0.10 3/3 4.42 M(3) S(3) 87 S S(90) 0.11 6/6 3.21 A(6) T(3) 85 W E(81) 0.16 35/6 2.93 T(3) A(6) L(3) W(3) H(3)

Table 4. The top 25% of residues in 1ezwA at the interface with 1ezwA1. (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. 6. Residues in 1ezwA, at the interface with 1ezwA1, colored by their relative importance. 1ezwA1 is shown in backbone representation (See Table 5. Appendix for the coloring scheme for the protein chain 1ezwA.) res type disruptive mutations 27 E (FWH)(YVCARG)(T)(SNKLPI) Table 6. continued 56 T (KR)(FQMWH)(NELPI)(D) res type subst’s cvg noc/ dist 89 G (KER)(FQMWHD)(NYLPI)(SVA) A˚ 90 R (TD)(SYEVCLAPIG)(FMW)(N) (%) bb ( ) 53 A (YR)(K)(H)(E) Y(3) 87 S (KR)(QH)(FMW)(E) D(6) 85 W (K)(E)(Q)(R) L(3) M(3) 78 S S(84) 0.14 42/13 2.77 Table 5. List of disruptive mutations for the top 25% of residues in M(3) 1ezwA, that are at the interface with 1ezwA1. R(6) A(3) Figure 6 shows residues in 1ezwA colored by their importance, at the Q(3) interface with 1ezwA1. 85 W E(81) 0.16 73/0 3.33 Interface with 1ezwA3.Table 6 lists the top 25% of residues at T(3) the interface with 1ezwA3. The following table (Table 7) suggests A(6) possible disruptive replacements for these residues (see Section 3.6). L(3) Table 6. W(3) res type subst’s cvg noc/ dist H(3) 107 G G(81) 0.19 20/20 3.52 (%) bb (A˚ ) A(12) 39 H H(87) 0.07 4/4 4.66 K(3) D(3) N(3) V(6) 69 Y Y(72) 0.21 6/6 3.38 S(3) Q(6) 40 Y Y(87) 0.09 92/22 2.64 F(15) P(6) N(3) H(3) V(3) T(3) 42 N N(84) 0.13 39/24 2.77 continued in next column Table 6. The top 25% of residues in 1ezwA at the interface with 1ezwA3. (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

4 contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 7. res type disruptive mutations 39 H (E)(Q)(KM)(TD) 40 Y (K)(Q)(M)(ER) 42 N (Y)(H)(R)(T) 78 S (YH)(R)(FKW)(E) 85 W (K)(E)(Q)(R) 107 G (E)(R)(FYWH)(KD) Fig. 8. A possible active surface on the chain 1ezwA. The larger cluster it 69 Y (K)(E)(R)(QM) belongs to is shown in blue.

Table 7. List of disruptive mutations for the top 25% of residues in Table 8. 1ezwA, that are at the interface with 1ezwA3. res type substitutions(%) cvg 27 E E(100) 0.04 56 T T(100) 0.04 89 G G(100) 0.04 90 R R(100) 0.04 53 A A(93)M(3)S(3) 0.10 87 S S(90)A(6)T(3) 0.11 85 W E(81)T(3)A(6) 0.16 L(3)W(3)H(3) 30 G G(84)A(3)D(12) 0.22

Table 8. Residues forming surface ”patch” in 1ezwA.

Table 9. res type disruptive mutations 27 E (FWH)(YVCARG)(T)(SNKLPI) 56 T (KR)(FQMWH)(NELPI)(D) 89 G (KER)(FQMWHD)(NYLPI)(SVA) 90 R (TD)(SYEVCLAPIG)(FMW)(N) 53 A (YR)(K)(H)(E) 87 S (KR)(QH)(FMW)(E) 85 W (K)(E)(Q)(R) 30 G (R)(K)(H)(E) Fig. 7. Residues in 1ezwA, at the interface with 1ezwA3, colored by their relative importance. 1ezwA3 is shown in backbone representation (See Table 9. Disruptive mutations for the surface patch in 1ezwA. Appendix for the coloring scheme for the protein chain 1ezwA.) Another group of surface residues is shown in Fig.9. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Figure 7 shows residues in 1ezwA colored by their importance, at the The residues belonging to this surface ”patch” are listed in Table 10, interface with 1ezwA3. while Table 11 suggests possible disruptive replacements for these 2.4.3 Possible novel functional surfaces at 25% coverage. One residues (see Section 3.6). group of residues is conserved on the 1ezwA surface, away from (or Table 10. susbtantially larger than) other functional sites and interfaces reco- res type substitutions(%) cvg gnizable in PDB entry 1ezw. It is shown in Fig. 8. The right panel 172 P P(100) 0.04 shows (in blue) the rest of the larger cluster this surface belongs to. 179 G G(100) 0.04 The residues belonging to this surface ”patch” are listed in Table 191 N D(96)N(3) 0.04 8, while Table 9 suggests possible disruptive replacements for these 329 G G(100) 0.04 residues (see Section 3.6). continued in next column

5 Table 10. continued res type substitutions(%) cvg 197 A A(78)G(12)M(6) 0.15 I(3) 252 I I(90)L(3)V(6) 0.15 177 A A(90)V(3)G(6) 0.17 10 L F(72)L(21).(6) 0.18 99 D D(84)N(3)H(6) 0.18 E(6) 244 A A(84).(6)E(3) 0.18 Q(3)R(3) 248 V V(75)P(12).(6) 0.18 Fig. 9. Another possible active surface on the chain 1ezwA. The larger cluster F(6) it belongs to is shown in blue. 198 S S(81)G(3)A(12) 0.19 E(3) Table 10. continued 251 F F(81)Y(6)R(6) 0.19 res type substitutions(%) cvg M(3)V(3) 64 G G(90)A(6)C(3) 0.05 97 P P(84)A(12)R(3) 0.20 67 N N(90)P(6)D(3) 0.06 182 M M(78)A(6)V(12) 0.20 98 G G(90)A(3)S(6) 0.06 T(3) 176 G G(90)A(9) 0.06 69 Y Y(72)Q(6)F(15) 0.21 200 P P(90)T(3)E(6) 0.06 N(3)V(3) 298 F F(90)I(3)L(6) 0.06 257 P P(81)A(12)D(3) 0.21 39 H H(87)D(3)V(6) 0.07 E(3) S(3) 65 I V(78)I(21) 0.23 194 L L(87)V(9)M(3) 0.07 127 P P(87)A(3).(9) 0.23 255 G G(90)H(6)A(3) 0.08 281 G G(72)P(6)F(12) 0.24 40 Y Y(87)P(6)H(3) 0.09 Y(6)M(3) T(3) 178 Q Q(78)K(3)L(6) 0.25 206 A A(87)H(6)F(3) 0.09 Y(3)E(6)N(3) N(3) 229 Y Y(87).(6)S(3) 0.09 Table 10. Residues forming surface ”patch” in 1ezwA. Q(3) 327 P P(90)V(3)D(3) 0.09 .(3) Table 11. 264 H H(84)A(3)S(9) 0.11 res type disruptive Y(3) mutations 326 S S(84)E(3)L(9) 0.11 172 P (YR)(TH)(SKECG)(FQWD) N(3) 179 G (KER)(FQMWHD)(NYLPI)(SVA) 9 E E(84)G(3).(6) 0.13 191 N (Y)(FWH)(TR)(VCAG) T(3)L(3) 329 G (KER)(FQMWHD)(NYLPI)(SVA) 42 N N(84)Y(3)D(6) 0.13 64 G (KER)(QHD)(FMW)(Y) L(3)M(3) 67 N (Y)(H)(FW)(TR) 180 P P(90)D(3)E(3) 0.13 98 G (KR)(E)(QH)(FMW) V(3) 176 G (KER)(QHD)(FYMW)(N) 196 N N(84)S(3)W(6) 0.13 200 P (R)(YH)(K)(T) Q(3)I(3) 298 F (KE)(T)(R)(QD) 202 D D(84)E(3)A(6) 0.13 39 H (E)(Q)(KM)(TD) L(3)T(3) 194 L (Y)(R)(H)(T) 263 R R(84)C(3)N(6) 0.13 255 G (E)(K)(R)(QD) H(3)G(3) 40 Y (K)(Q)(M)(ER) 174 Y Y(90)L(6)H(3) 0.14 206 A (E)(K)(YR)(D) 283 F F(84)T(3)Y(9) 0.14 229 Y (K)(M)(Q)(VLAPIR) W(3) 327 P (R)(Y)(H)(T) continued in next column 264 H (E)(Q)(KM)(D) continued in next column

6 Table 11. continued guide - due to rounding errors these percentages often do not add up res type disruptive to 100%. mutations 326 S (R)(H)(FKW)(Y) 3.3 Surface 9 E (H)(FW)(R)(Y) To detect candidates for novel functional interfaces, first we look for 42 N (Y)(H)(R)(T) residues that are solvent accessible (according to DSSP program) by 2 180 P (R)(Y)(H)(T) at least 10A˚ , which is roughly the area needed for one water mole- 196 N (Y)(T)(H)(FWR) cule to come in the contact with the residue. Furthermore, we require 202 D (R)(H)(FW)(KY) that these residues form a “cluster” of residues which have neighbor 263 R (D)(E)(T)(Y) within 5A˚ from any of their heavy atoms. 174 Y (K)(Q)(M)(E) Note, however, that, if our picture of protein evolution is correct, 283 F (K)(E)(Q)(D) the neighboring residues which are not surface accessible might be 197 A (YR)(KE)(H)(D) equally important in maintaining the interaction specificity - they 252 I (YR)(H)(T)(KE) should not be automatically dropped from consideration when choo- 177 A (KER)(Y)(QHD)(N) sing the set for mutagenesis. (Especially if they form a cluster with 10 L (R)(Y)(T)(H) the surface residues.) 99 D (R)(FW)(H)(YVCAG) 3.4 Number of contacts 244 A (Y)(H)(R)(E) 248 V (KER)(Y)(QD)(H) Another column worth noting is denoted “noc/bb”; it tells the num- 198 S (R)(K)(H)(FW) ber of contacts heavy atoms of the residue in question make across 251 F (E)(K)(D)(T) the interface, as well as how many of them are realized through the 97 P (Y)(T)(R)(H) backbone atoms (if all or most contacts are through the backbone, 182 M (Y)(H)(R)(T) mutation presumably won’t have strong impact). Two heavy atoms 69 Y (K)(E)(R)(QM) are considered to be “in contact” if their centers are closer than 5A˚ . 257 P (R)(Y)(H)(T) 3.5 Annotation 65 I (YR)(H)(TKE)(SQCDG) 127 P (Y)(R)(H)(T) If the residue annotation is available (either from the pdb file or 281 G (KR)(E)(QD)(H) from other sources), another column, with the header “annotation” 178 Q (Y)(FW)(TH)(VCAG) 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 Table 11. Disruptive mutations for the surface patch in 1ezwA. bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 3.1 Coverage they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For 4 APPENDIX 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- 4.1 File formats sely, when looking for substitutions which will not affect the protein, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment • is given in the immediately following bracket. No percentage is given alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • residue# residue number in the PDB file

7 seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, COVERAGE and freely distributed under the GNU General Public License. 4.3.2 CE To map ligand binding sites from different V source structures, report maker uses the CE program: 100% 50% 30% 5% http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 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, V which is roughly the area needed for one water molecule to come in RELATIVE IMPORTANCE the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, Fig. 10. Coloring scheme used to color residues by their relative importance. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type gnment as a starting point for the analysis (sequences shorter than • rank rank of the position according to older version of ET 75% of the query are taken out, however); R. Schneider, A. de • variability has two subfields: Daruvar, and C. Sander. ”The HSSP database of protein structure- 1. number of different amino acids appearing in in this column sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of the alignment http://swift.cmbi.kun.nl/swift/hssp/ 2. their type 4.3.5 LaTex The text for this report was processed using LAT X; • E rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 10. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version

8 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1ezwA.etvx - ET viewer input file for 1ezwA tionary Trace Method Defines Binding Surfaces Common to Protein • 1ezwA.cluster report.summary - Cluster report summary for Families” J. Mol. Bio. 257: 342-358. 1ezwA report maker itself is described in Mihalek I., I. Res and O. • 1ezwA.ranks - Ranks file in sequence order for 1ezwA Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics • 1ezwA.clusters - Cluster descriptions for 1ezwA 22:1656-7. • 1ezwA.msf - the multiple sequence alignment used for the chain 1ezwA 4.6 About report maker • 1ezwA.descr - description of sequences used in 1ezwA msf report maker was written in 2006 by Ivana Mihalek. The 1D ran- • king visualization program was written by Ivica Res.ˇ report maker 1ezwA.ranks sorted - full listing of residues and their ranking is copyrighted by Lichtarge Lab, Baylor College of Medicine, for 1ezwA Houston. • 1ezwA.1ezwAMG361.if.pml - Pymol script for Figure 5 • 4.7 Attachments 1ezwA.cbcvg - used by other 1ezwA – related pymol scripts • The following files should accompany this report: 1ezwA.1ezwA1.if.pml - Pymol script for Figure 6 • 1ezwA.1ezwA3.if.pml - Pymol script for Figure 7 • 1ezwA.complex.pdb - coordinates of 1ezwA with all of its interacting partners

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