Pages 1–8 3bwh Evolutionary trace report by report maker June 14, 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 3bwh): Title: Atomic resolution structure of cucurmosin, a novel type 1 rip from the sarcocarp of cucurbita moschata Compound: Mol id: 1; molecule: cucurmosin; chain: a Organism, scientific name: Cucurbita Moschata; 3bwh contains a single unique chain 3bwhA (244 residues long).

2 CHAIN 3BWHA CONTENTS 2.1 P29339 overview 1 Introduction 1 From SwissProt, id P29339, 63% identical to 3bwhA: Description: Ribosome-inactivating protein momordin II precursor 2 Chain 3bwhA 1 (EC 3.2.2.22) (rRNA N-glycosidase). 2.1 P29339 overview 1 Organism, scientific name: balsamina (Bitter gourd) 2.2 Multiple sequence alignment for 3bwhA 1 (Balsam apple). 2.3 Residue ranking in 3bwhA 1 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.4 Top ranking residues in 3bwhA and their position on Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core the structure 1 eudicotyledons; ; eurosids I; ; ; 2.4.1 Clustering of residues at 25% coverage. 2 Momordica. 2.4.2 Overlap with known functional surfaces at Catalytic activity: Endohydrolysis of the N-glycosidic bond at one 25% coverage. 2 specific adenosine on the 28S rRNA. 2.4.3 Possible novel functional surfaces at 25% Similarity: Belongs to the ribosome-inactivating protein family. coverage. 5 Type 1 RIP subfamily. About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 6 collaboration between the Swiss Institute of Bioinformatics and the 3.1 Coverage 6 EMBL outstation - the European Bioinformatics Institute. There are 3.2 Known substitutions 6 no restrictions on its use as long as its content is in no way modified 3.3 Surface 6 and this statement is not removed. 3.4 Number of contacts 6 3.5 Annotation 7 2.2 Multiple sequence alignment for 3bwhA 3.6 Mutation suggestions 7 For the chain 3bwhA, the alignment 3bwhA.msf (attached) with 86 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 3bwhA.msf. Its statistics, from the 4.3 Credits 7 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-122 in 3bwhA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 2. Residues 123-244 in 3bwhA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 3. Residues in 3bwhA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 86 Total number of residues: 20242 Smallest: 202 Largest: 244 Average length: 235.4 Alignment length: 244 Average identity: 36% Most related pair: 99% Most unrelated pair: 17% Most distant seq: 30%

Furthermore, 1% of residues show as conserved in this alignment. The alignment consists of 76% eukaryotic ( 76% plantae) sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 3bwhA.descr. 2.3 Residue ranking in 3bwhA The 3bwhA sequence is shown in Figs. 1–2, with each residue colo- red according to its estimated importance. The full listing of residues in 3bwhA can be found in the file called 3bwhA.ranks sorted in the attachment. 2.4 Top ranking residues in 3bwhA and their position on Fig. 4. Residues in 3bwhA, colored according to the cluster they belong to: the structure red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The In the following we consider residues ranking among top 25% of corresponding Pymol script is attached. residues in the protein . Figure 3 shows residues in 3bwhA colored by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A in Table 1. Pymol script for producing this figure can be found in the attachment.

2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.4 are composed of the residues listed

2 Table 1. cluster size member color residues red 58 4,14,17,21,22,25,34,35,36,37 47,52,63,65,66,68,70,71,72 73,74,81,82,103,105,107,109 112,116,120,123,125,126,130 134,146,148,150,153,154,158 159,160,161,162,163,164,165 179,186,187,188,190,193,194 197,198,200

Table 1. Clusters of top ranking residues in 3bwhA.

2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. 1,2-ethanediol binding site. Table 2 lists the top 25% of residues at the interface with 3bwhAEDO257 (1,2-ethanediol). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Fig. 5. Residues in 3bwhA, at the interface with 1,2-ethanediol, colored by Table 2. their relative importance. The ligand (1,2-ethanediol) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on res type subst’s cvg noc/ dist the line of sight to the ligand were removed. (See Appendix for the coloring ˚ (%) bb (A) scheme for the protein chain 3bwhA.) 35 P P(91) 0.07 7/1 3.94 T(2) M(1) 1,2-ethanediol binding site. Table 4 lists the top 25% of residues I(1) at the interface with 3bwhAEDO255 (1,2-ethanediol). The following N(1) table (Table 5) suggests possible disruptive replacements for these R(1) residues (see Section 3.6). L(1) 25 L V(26) 0.19 1/1 4.54 Table 4. L(52) res type subst’s cvg noc/ dist A(15) (%) bb (A˚ ) F(4) 35 P P(91) 0.07 3/1 3.70 I(1) T(2) M(1) Table 2. The top 25% of residues in 3bwhA at the interface with 1,2- I(1) ethanediol.(Field names: res: residue number in the PDB entry; type: amino N(1) acid type; substs: substitutions seen in the alignment; with the percentage of R(1) each type in the bracket; noc/bb: number of contacts with the ligand, with L(1) the number of contacts realized through backbone atoms given in the bracket; 34 I I(80) 0.19 11/6 3.79 dist: distance of closest apporach to the ligand. ) L(15) F(1) T(1) Table 3. G(1) res type disruptive V(1) mutations 35 P (Y)(R)(H)(T) Table 4. The top 25% of residues in 3bwhA at the interface with 1,2- 25 L (R)(Y)(TKEH)(SQCDG) ethanediol.(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 Table 3. List of disruptive mutations for the top 25% of residues in the number of contacts realized through backbone atoms given in the bracket; 3bwhA, that are at the interface with 1,2-ethanediol. dist: distance of closest apporach to the ligand. )

Figure 5 shows residues in 3bwhA colored by their importance, at the interface with 3bwhAEDO257.

3 Table 5. Table 6. continued res type disruptive res type subst’s cvg noc/ dist antn mutations (%) bb (A˚ ) 35 P (Y)(R)(H)(T) 153 I I(95) 0.05 8/0 3.75 34 I (R)(Y)(K)(H) L(2) V(1) Table 5. List of disruptive mutations for the top 25% of residues in A(1) 3bwhA, that are at the interface with 1,2-ethanediol. 107 G G(75) 0.12 6/6 2.77 site S(20) T(1) E(1) N(1)

Table 6. The top 25% of residues in 3bwhA at the interface with 1,2- ethanediol.(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 70 Y (K)(Q)(EMR)(N) 109 Y (K)(QM)(NEVLAPIR)(D) 158 E (H)(FYWR)(CG)(TKVA) 161 R (T)(YD)(SECG)(VA) 153 I (YR)(H)(TKE)(SQCDG) 107 G (R)(K)(FWH)(E)

Table 7. List of disruptive mutations for the top 25% of residues in 3bwhA, that are at the interface with 1,2-ethanediol. Fig. 6. Residues in 3bwhA, at the interface with 1,2-ethanediol, colored by their relative importance. The ligand (1,2-ethanediol) is colored green. Atoms Figure 7 shows residues in 3bwhA colored by their importance, at the A further than 30 ˚ away from the geometric center of the ligand, as well as on interface with 3bwhAEDO253. the line of sight to the ligand were removed. (See Appendix for the coloring 1,2-ethanediol binding site. Table 8 lists the top 25% of residues scheme for the protein chain 3bwhA.) at the interface with 3bwhAEDO256 (1,2-ethanediol). The following table (Table 9) suggests possible disruptive replacements for these Figure 6 shows residues in 3bwhA colored by their importance, at the residues (see Section 3.6). interface with 3bwhAEDO255. Table 8. 1,2-ethanediol binding site. Table 6 lists the top 25% of residues res type subst’s cvg noc/ dist antn at the interface with 3bwhAEDO253 (1,2-ethanediol). The following (%) bb (A˚ ) table (Table 7) suggests possible disruptive replacements for these 163 R N(10) 0.21 15/9 3.72 residues (see Section 3.6). K(47) Table 6. R(34) res type subst’s cvg noc/ dist antn T(3) (%) bb (A˚ ) E(1) 70 Y Y(98) 0.02 12/1 4.01 Q(2) A(1) 164 Y P(10) 0.24 22/10 3.45 site 109 Y Y(100) 0.02 18/1 3.71 Y(61) 158 E E(98) 0.03 4/0 3.85 F(17) A(1) A(5) 161 R R(98) 0.03 7/0 3.06 site I(1) L(1) H(1) continued in next column continued in next column

4 Fig. 7. Residues in 3bwhA, at the interface with 1,2-ethanediol, colored by Fig. 8. Residues in 3bwhA, at the interface with 1,2-ethanediol, colored by their relative importance. The ligand (1,2-ethanediol) is colored green. Atoms their relative importance. The ligand (1,2-ethanediol) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on 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 the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 3bwhA.) scheme for the protein chain 3bwhA.)

Table 8. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) R(1) C(1)

Table 8. The top 25% of residues in 3bwhA at the interface with 1,2- ethanediol.(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. 9. A possible active surface on the chain 3bwhA. The larger cluster it belongs to is shown in blue.

Table 9. res type disruptive The residues belonging to this surface ”patch” are listed in Table 10, mutations while Table 11 suggests possible disruptive replacements for these 163 R (TY)(FVAWD)(CG)(S) residues (see Section 3.6). 164 Y (K)(EQ)(M)(R) Table 10. res type substitutions(%) cvg antn Table 9. List of disruptive mutations for the top 25% of residues in 70 Y Y(98)A(1) 0.02 3bwhA, that are at the interface with 1,2-ethanediol. 109 Y Y(100) 0.02 190 W W(100) 0.02 Figure 8 shows residues in 3bwhA colored by their importance, at the 158 E E(98)A(1) 0.03 interface with 3bwhAEDO256. 161 R R(98)L(1) 0.03 site 22 R R(95)Q(4) 0.04 2.4.3 Possible novel functional surfaces at 25% coverage. One 153 I I(95)L(2)V(1) 0.05 group of residues is conserved on the 3bwhA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- continued in next column gnizable in PDB entry 3bwh. It is shown in Fig. 9. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

5 Table 10. continued Table 10. continued res type substitutions(%) cvg antn res type substitutions(%) cvg antn A(1) R(1)C(1) 179 P P(91)C(1)V(2) 0.06 F(1)A(2)N(1) Table 10. Residues forming surface ”patch” in 3bwhA. 120 R R(96)G(1)I(1) 0.07 K(1) 186 L L(82)M(11)Y(2) 0.08 F(2)I(1) Table 11. 74 Y Y(83)F(15)C(1) 0.09 res type disruptive 146 A A(89)P(4)K(2) 0.09 mutations R(2)N(1) 70 Y (K)(Q)(EMR)(N) 213 L L(91)I(5)S(1) 0.11 109 Y (K)(QM)(NEVLAPIR)(D) .(1) 190 W (KE)(TQD)(SNCRG)(M) 107 G G(75)S(20)T(1) 0.12 site 158 E (H)(FYWR)(CG)(TKVA) E(1)N(1) 161 R (T)(YD)(SECG)(VA) 103 L L(74)I(16)F(5) 0.13 22 R (T)(YD)(SVCAG)(FELWPI) N(1)T(1)Y(1) 153 I (YR)(H)(TKE)(SQCDG) 68 N N(86)S(4)D(8) 0.14 179 P (R)(Y)(H)(E) T(1) 120 R (TYD)(E)(S)(FVCAWG) 73 A A(50)G(50) 0.14 186 L (R)(Y)(T)(K) 188 N T(16)N(67)E(12) 0.15 74 Y (K)(Q)(M)(E) A(1)L(1)K(1) 146 A (Y)(E)(HDR)(K) 65 D D(68)R(13)N(11) 0.16 213 L (R)(Y)(H)(T) T(2)S(1)Q(1) 107 G (R)(K)(FWH)(E) A(1) 103 L (R)(Y)(KH)(TE) 47 F F(50)Y(41)L(5) 0.17 68 N (FYWH)(R)(TEVMA)(KCG) H(1)A(1) 73 A (KER)(Y)(QHD)(N) 123 I I(66)V(23)L(4) 0.17 188 N (Y)(H)(FW)(T) T(2)F(2)A(1) 65 D (R)(FWH)(Y)(K) 81 Y Y(82)F(9)H(2) 0.18 47 F (E)(K)(Q)(D) V(3)A(1)N(1) 123 I (R)(Y)(K)(H) 105 Y F(59)Y(37)V(2) 0.18 81 Y (K)(EQ)(M)(R) C(1) 105 Y (K)(Q)(E)(MR) 25 L V(26)L(52)A(15) 0.19 25 L (R)(Y)(TKEH)(SQCDG) F(4)I(1) 82 F (E)(K)(Q)(D) 82 F Y(3)F(72)T(1) 0.20 163 R (TY)(FVAWD)(CG)(S) H(12)I(2)L(6) 72 V (YR)(KE)(H)(D) R(1) 116 A (R)(KY)(H)(E) 163 R N(10)K(47)R(34) 0.21 71 I (R)(Y)(KH)(TE) T(3)E(1)Q(2) 148 A (R)(K)(E)(Y) 72 V V(60)M(27)I(4) 0.22 187 E (Y)(H)(FW)(CRG) L(3)A(2)W(1) 164 Y (K)(EQ)(M)(R) 116 A A(83)V(4)G(4) 0.22 .(4)E(2) Table 11. Disruptive mutations for the surface patch in 3bwhA. 71 I V(72)I(16)Y(1) 0.23 L(10) 148 A S(33)A(24)G(3) 0.23 F(23)L(1)T(9) 3 NOTES ON USING TRACE RESULTS P(4) 187 E E(87)K(1)Q(9) 0.23 3.1 Coverage V(1)I(1) Trace results are commonly expressed in terms of coverage: the resi- 164 Y P(10)Y(61)F(17) 0.24 site due is important if its “coverage” is small - that is if it belongs to A(5)I(1)H(1) some small top percentage of residues [100% is all of the residues continued in next column 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 pressure on the residue.) Starting from the top of that list, mutating a

6 couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3.2 Known substitutions

One of the table columns is “substitutions” - other amino acid types COVERAGE seen at the same position in the alignment. These amino acid types

may be interchangeable at that position in the protein, so if one wants V to affect the protein by a point mutation, they should be avoided. For 100% 50% 30% 5% 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 is given in the immediately following bracket. No percentage is given V in the cases when it is smaller than 1%. This is meant to be a rough RELATIVE IMPORTANCE guide - due to rounding errors these percentages often do not add up to 100%. Fig. 10. Coloring scheme used to color residues by their relative importance. 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 acid, and they are grouped in round brackets if they appear equally 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- sing the set for mutagenesis. (Especially if they form a cluster with 4 APPENDIX the surface residues.) 4.1 File formats 3.4 Number of contacts Files with extension “ranks sorted” are the actual trace results. The 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 backbone atoms (if all or most contacts are through the backbone, • residue# residue number in the PDB file mutation presumably won’t have strong impact). Two heavy atoms • type amino acid type ˚ are considered to be “in contact” if their centers are closer than 5A. • rank rank of the position according to older version of ET 3.5 Annotation • variability has two subfields: If the residue annotation is available (either from the pdb file or 1. number of different amino acids appearing in in this column from other sources), another column, with the header “annotation” of the alignment appears. Annotations carried over from PDB are the following: site 2. their type (indicating existence of related site record in PDB ), S-S (disulfide • rho ET score - the smaller this value, the lesser variability of bond forming residue), hb (hydrogen bond forming residue, jb (james this position across the branches of the tree (and, presumably, bond forming residue), and sb (for salt bridge forming residue). the greater the importance for the protein) • 3.6 Mutation suggestions cvg coverage - percentage of the residues on the structure which have this rho or smaller Mutation suggestions are completely heuristic and based on comple- • mentarity with the substitutions found in the alignment. Note that gaps percentage of gaps in this column they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following 4.2 Color schemes used properties: small [AV GSTC], medium [LPNQDEMIK], large The following color scheme is used in figures with residues colored [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by cluster size: black is a single-residue cluster; clusters composed of tively [KHR], or negatively [DE] charged, aromatic [WFYH], more than one residue colored according to this hierarchy (ordered long aliphatic chain [EKRQM], OH-group possession [SDETY ], by descending size): red, blue, yellow, green, purple, azure, tur- and NH2 group possession [NQRK]. The suggestions are listed quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, according to how different they appear to be from the original amino bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine,

7 DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, http://pymol.sourceforge.net/. (Note for Windows tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. users: the attached package needs to be unzipped for Pymol to read The colors used to distinguish the residues by the estimated the scripts and launch the viewer.) evolutionary pressure they experience can be seen in Fig. 10. 4.4 Note about ET Viewer 4.3 Credits Dan Morgan from the Lichtarge lab has developed a visualization 4.3.1 Alistat alistat reads a multiple sequence alignment from the tool specifically for viewing trace results. If you are interested, please file and shows a number of simple statistics about it. These stati- visit: stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the http://mammoth.bcm.tmc.edu/traceview/ alignment length (e.g. including gap characters). Also shown are The viewer is self-unpacking and self-installing. Input files to be used some percent identities. A percent pairwise alignment identity is defi- with ETV (extension .etvx) can be found in the attachment to the ned as (idents / MIN(len1, len2)) where idents is the number of main report. exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and 4.5 Citing this work ”most unrelated pair” of the alignment are the average, maximum, The method used to rank residues and make predictions in this report and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of seq” is calculated by finding the maximum pairwise identity (best Evolution-Entropy Hybrid Methods for Ranking of Protein Residues relative) for all N sequences, then finding the minimum of these N by Importance” J. Mol. Bio. 336: 1265-82. For the original version numbers (hence, the most outlying sequence). alistat is copyrighted of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- by HHMI/Washington University School of Medicine, 1992-2001, tionary Trace Method Defines Binding Surfaces Common to Protein and freely distributed under the GNU General Public License. Families” J. Mol. Bio. 257: 342-358. 4.3.2 CE To map ligand binding sites from different report maker itself is described in Mihalek I., I. Res and O. source structures, report maker uses the CE program: Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) of service for comparative analysis of proteins.” Bioinformatics ”Protein structure alignment by incremental combinatorial extension 22:1656-7. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.6 About report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker was written in 2006 by Ivana Mihalek. The 1D ran- 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , king visualization program was written by Ivica Res.ˇ report maker which is roughly the area needed for one water molecule to come in is copyrighted by Lichtarge Lab, Baylor College of Medicine, the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Houston. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, 4.7 Attachments http://www.cmbi.kun.nl/gv/dssp/descrip.html. The following files should accompany this report: • 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 3bwhA.complex.pdb - coordinates of 3bwhA with all of its gnment as a starting point for the analysis (sequences shorter than interacting partners 75% of the query are taken out, however); R. Schneider, A. de • 3bwhA.etvx - ET viewer input file for 3bwhA Daruvar, and C. Sander. ”The HSSP database of protein structure- • 3bwhA.cluster report.summary - Cluster report summary for sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 3bwhA http://swift.cmbi.kun.nl/swift/hssp/ • 3bwhA.ranks - Ranks file in sequence order for 3bwhA • 3bwhA.clusters - Cluster descriptions for 3bwhA 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 3bwhA.msf - the multiple sequence alignment used for the chain Wesley,” Reading, Mass. (1986). 3bwhA • 4.3.6 Muscle When making alignments “from scratch”, report 3bwhA.descr - description of sequences used in 3bwhA msf maker uses Muscle alignment program: Edgar, Robert C. (2004), • 3bwhA.ranks sorted - full listing of residues and their ranking ”MUSCLE: multiple sequence alignment with high accuracy and for 3bwhA high throughput.” Nucleic Acids Research 32(5), 1792-97. • 3bwhA.3bwhAEDO257.if.pml - Pymol script for Figure 5 http://www.drive5.com/muscle/ • 3bwhA.cbcvg - used by other 3bwhA – related pymol scripts • 4.3.7 Pymol The figures in this report were produced using 3bwhA.3bwhAEDO255.if.pml - Pymol script for Figure 6 Pymol. The scripts can be found in the attachment. Pymol • 3bwhA.3bwhAEDO253.if.pml - Pymol script for Figure 7 is an open-source application copyrighted by DeLano Scien- • 3bwhA.3bwhAEDO256.if.pml - Pymol script for Figure 8 tific LLC (2005). For more information about Pymol see

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