2Aip Lichtarge Lab 2006

Pages 1–8 2aip Evolutionary trace report by report maker October 9, 2009

4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 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 2aip): Title: Crystal structure of native protein c activator from the venom of copperhead snake agkistrodon contortrix contortrix Compound: Mol id: 1; molecule: protein c activator; chain: a; synonym: venombin a, ancrod, acc-c; ec: 3.4.21.74 Organism, scientific name: Agkistrodon Contortrix Contortrix; 2aip contains a single unique chain 2aipA (231 residues long). CONTENTS 2 CHAIN 2AIPA 1 Introduction 1 2.1 P09872 overview 2 Chain 2aipA 1 From SwissProt, id P09872, 100% identical to 2aipA: 2.1 P09872 overview 1 Description: Ancrod (EC 3.4.21.74) (Venombin A) (Protein C 2.2 Multiple sequence alignment for 2aipA 1 activator) (ACC-C). 2.3 Residue ranking in 2aipA 1 Organism, scientific name: Agkistrodon contortrix contortrix 2.4 Top ranking residues in 2aipA and their position on (Southern copperhead). the structure 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.4.1 Clustering of residues at 25% coverage. 2 Euteleostomi; Lepidosauria; Squamata; Scleroglossa; Serpentes; 2.4.2 Overlap with known functional surfaces at Colubroidea; Viperidae; Crotalinae; Agkistrodon. 25% coverage. 2 Function: Thrombin-like snake venom serine protease. Cleaves 2.4.3 Possible novel functional surfaces at 25% fibrinopeptides AM, AO, and AY; the aberrant fibrinogen is then coverage. 5 incapable of being cross-linked, forming easily dispersible clots. Activates protein C. 3 Notes on using trace results 6 Catalytic activity: Selective cleavage of Arg-—-Xaa bond in fibri- 3.1 Coverage 6 nogen, to form fibrin, and release fibrinopeptide A. The specificity 3.2 Known substitutions 6 of further degradation of fibrinogen varies with species origin of the 3.3 Surface 6 enzyme. 3.4 Number of contacts 6 Subcellular location: Secreted (Potential). 3.5 Annotation 7 Tissue specificity: Expressed by the venom gland. 3.6 Mutation suggestions 7 Similarity: Belongs to the peptidase S1 family. Snake venom subfamily. 4 Appendix 7 Similarity: Contains 1 peptidase S1 domain. 4.1 File formats 7 About: This Swiss-Prot entry is copyright. It is produced through a 4.2 Color schemes used 7 collaboration between the Swiss Institute of Bioinformatics and the 4.3 Credits 7 EMBL outstation - the European Bioinformatics Institute. There are

1 Lichtarge lab 2006 Fig. 1. Residues 16-134 in 2aipA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.) Note that some residues in 2aipA carry insertion code. Fig. 2. Residues 135-245 in 2aipA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.) Note that some residues in 2aipA carry insertion code. no restrictions on its use as long as its content is in no way modiﬁed and this statement is not removed.

2.2 Multiple sequence alignment for 2aipA For the chain 2aipA, the alignment 2aipA.msf (attached) with 1080 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 2aipA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 1080 Total number of residues: 238093 Smallest: 174 Largest: 231 Average length: 220.5 Alignment length: 231 Average identity: 35% Most related pair: 99% Most unrelated pair: 15% Most distant seq: 37%

Fig. 3. Residues in 2aipA, colored by their relative importance. Clockwise: Furthermore, <1% of residues show as conserved in this ali- front, back, top and bottom views. gnment. The alignment consists of 49% eukaryotic ( 38% vertebrata, 10% arthropoda, <1% fungi), <1% prokaryotic, and <1% viral 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the sequences. (Descriptions of some sequences were not readily availa- top 25% of all residues, this time colored according to clusters they ble.) The file containing the sequence descriptions can be found in belong to. The clusters in Fig.4 are composed of the residues listed the attachment, under the name 2aipA.descr. in Table 1. Table 1. 2.3 Residue ranking in 2aipA cluster size member The 2aipA sequence is shown in Figs. 1–2, with each residue colored color residues according to its estimated importance. The full listing of residues red 54 42,43,44,46,51,52,54,55,56 in 2aipA can be found in the file called 2aipA.ranks sorted in the 57,58,102,103,104,105,108 attachment. 123,124,136,139,140,141,142 155,168,180,182,183,184,186 2.4 Top ranking residues in 2aipA and their position on 189,191,193,194,195,196,197 the structure 198,199,211,213,214,215,216 220,221,225,226,227,228,229 In the following we consider residues ranking among top 25% of resi- 231,237,238 dues in the protein . Figure 3 shows residues in 2aipA colored by their importance: bright red and yellow indicate more conserved/important continued in next column residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

2 Table 3. res type disruptive mutations 91 C (KER)(Q)(MD)(FWH) 237 W (K)(E)(Q)(D)

Table 3. List of disruptive mutations for the top 25% of residues in 2aipA, that are at the interface with sulfate ion.

Fig. 4. Residues in 2aipA, 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 blue 2 18,19

Table 1. Clusters of top ranking residues in 2aipA. Fig. 5. Residues in 2aipA, at the interface with sulfate ion, colored by their relative importance. The ligand (sulfate ion) 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 2.4.2 Overlap with known functional surfaces at 25% coverage. for the protein chain 2aipA.) The name of the ligand is composed of the source PDB identiﬁer and the heteroatom name used in that ﬁle. Sulfate ion binding site. Table 2 lists the top 25% of residues at the Figure 5 shows residues in 2aipA colored by their importance, at the interface with 2aipSO4303 (sulfate ion). The following table (Table interface with 2aipSO4303. 3) suggests possible disruptive replacements for these residues (see Sulfate ion binding site. Table 4 lists the top 25% of residues at the Section 3.6). interface with 2aipSO4301 (sulfate ion). The following table (Table 5) suggests possible disruptive replacements for these residues (see Table 2. Section 3.6). res type subst’s cvg noc/ dist antn Table 4. A˚ (%) bb ( ) res type subst’s cvg noc/ dist antn 91 C 228.(2) 0.03 15/10 3.32 S-S (%) bb (A˚ ) 35 194 D D(99)EV 0.01 3/3 4.05 237 W W(95) 0.08 1/0 4.77 . F(2)Y 42 C C(98)WV 0.03 1/0 4.30 S-S .(1)CSV .GA 58 C C(98)L. 0.03 1/0 5.00 S-S Table 2. The top 25% of residues in 2aipA at the interface with sulfate NVSA ion.(Field names: res: residue number in the PDB entry; type: amino acid 191 C C(96) 0.04 2/2 4.04 S-S 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- continued in next column ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

3 Table 4. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) F(2)YSG .A 195 S S(98)NG 0.05 14/5 2.87 FYPA. 57 H H(97)AR 0.07 12/0 2.75 QN.LSP 193 G G(92) 0.09 9/9 2.96 A(1) R(1)VSM QDYEFCH T.

Table 4. The top 25% of residues in 2aipA at the interface with sulfate ion.(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 2aipA, at the interface with sulfate ion, colored by their Table 5. relative importance. The ligand (sulfate ion) is colored green. Atoms further res type disruptive than 30A˚ away from the geometric center of the ligand, as well as on the line mutations of sight to the ligand were removed. (See Appendix for the coloring scheme 194 D (R)(H)(FW)(Y) for the protein chain 2aipA.) 42 C (K)(E)(R)(Q) 58 C (R)(K)(E)(H) 191 C (K)(R)(E)(Q) 195 S (KR)(Q)(H)(EM) 57 H (E)(T)(D)(Q) 193 G (KR)(E)(H)(Q)

Table 5. List of disruptive mutations for the top 25% of residues in 2aipA, that are at the interface with sulfate ion.

Figure 6 shows residues in 2aipA colored by their importance, at the interface with 2aipSO4301. Glycerol binding site. Table 6 lists the top 25% of residues at the interface with 2aipGOL401 (glycerol). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6). Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 56 R A(91) 0.11 15/0 2.76 G(4)V S(1)KLQ YTC.ERF

Table 6. The top 25% of residues in 2aipA at the interface with glycerol.(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. )

4 Table 7. Table 9. res type disruptive res type disruptive mutations mutations 56 R (D)(TYE)(LPI)(SFVCAWG) 148 N (Y)(FTW)(H)(R)

Table 7. List of disruptive mutations for the top 25% of residues in 2aipA, Table 9. List of disruptive mutations for the top 25% of residues in 2aipA, that are at the interface with glycerol. that are at the interface with NAG.

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

Figure 7 shows residues in 2aipA colored by their importance, at the Figure 8 shows residues in 2aipA colored by their importance, at the interface with 2aipGOL401. interface with 2aipANAG601. NAG binding site. Table 8 lists the top 25% of residues at the interface with 2aipANAG601 (nag). The following table (Table 9) 2.4.3 Possible novel functional surfaces at 25% coverage. One suggests possible disruptive replacements for these residues (see group of residues is conserved on the 2aipA surface, away from (or Section 3.6). susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2aip. It is shown in Fig. 9. The right panel Table 8. shows (in blue) the rest of the larger cluster this surface belongs to. res type subst’s cvg noc/ dist The residues belonging to this surface ”patch” are listed in Table 10, (%) bb (A˚ ) while Table 11 suggests possible disruptive replacements for these 148 N 204.(1) 0.01 45/4 1.44 residues (see Section 3.6). 66 Table 10. Table 8. The top 25% of residues in 2aipA at the interface with res type substitutions(%) cvg antn NAG.(Field names: res: residue number in the PDB entry; type: amino acid 198 P P(98)AH.S 0.01 type; substs: substitutions seen in the alignment; with the percentage of each 140 G G(99)ITVA 0.02 type in the bracket; noc/bb: number of contacts with the ligand, with the num- 91 C 228.(2)35 0.03 S-S ber of contacts realized through backbone atoms given in the bracket; dist: 141 W W(97)YF(1)ST 0.04 distance of closest apporach to the ligand. ) 191 C C(96)F(2)YSG.A 0.04 S-S continued in next column

5 Table 10. continued res type substitutions(%) cvg antn DT(1)CS(1)RI.QL XPVK 124 P A(8)P(80)G(1)RT 0.23 EVS(1)Q(1)NL(1) IWYCFDMK 180 T M(62)KT(12)N(6) 0.23 GQ(6)F(1)V(1) I(2)H(1)SE(2)AW LYP 139 M T(26)S(34)A(18) 0.24 Fig. 9. A possible active surface on the chain 2aipA. The larger cluster it L(1)M(12)YI(3) belongs to is shown in blue. V(1)QFHR

Table 10. continued Table 10. Residues forming surface ”patch” in 2aipA. res type substitutions(%) cvg antn 195 S S(98)NGFYPA. 0.05 197 G G(97)S(2)A.T 0.05 Table 11. 220 C C(95).(2)GTVHRW 0.06 S-S res type disruptive 57 H H(97)ARQN.LSP 0.07 mutations 182 C C(98)ILMVS 0.07 S-S 198 P (R)(Y)(K)(TE) 237 W W(95)F(2)Y.(1)C 0.08 140 G (R)(K)(E)(H) SV 91 C (KER)(Q)(MD)(FWH) 193 G G(92)A(1)R(1)VS 0.09 141 W (K)(E)(Q)(D) MQDYEFCHT. 191 C (K)(R)(E)(Q) 225 P P(89)Y(4)NAF(2) 0.10 195 S (KR)(Q)(H)(EM) TVS.LI 197 G (KR)(E)(Q)(H) 56 R A(91)G(4)VS(1)K 0.11 220 C (E)(K)(R)(D) LQYTC.ERF 57 H (E)(T)(D)(Q) 189 D D(83)A(3)S(5) 0.11 182 C (R)(K)(E)(H) G(5)N(1)QYET.C 237 W (K)(E)(Q)(D) 214 S T(1)S(90)NV(2) 0.12 193 G (KR)(E)(H)(Q) A(1)F(1)GHYQIWL 225 P (R)(Y)(H)(K) PXC 56 R (D)(TYE)(LPI)(SFVCAWG) 216 G WG(90)S(2)V(3)A 0.13 189 D (R)(H)(FW)(K) REITHPNXC 214 S (R)(K)(Q)(EH) 104 M A(48)G(2)M(37) 0.17 216 G (R)(E)(K)(H) S(5)L(2)TQV(1)K 104 M (Y)(H)(T)(R) .CXRPIH 238 I (R)(Y)(H)(T) 238 I I(85)L(5)M(1) 0.19 213 L (R)(Y)(H)(K) V(5)A.(1)FG 215 V (E)(Y)(R)(K) 213 L I(4)V(67)A(2) 0.20 227 I (R)(Y)(H)(T) T(19)F(1)DMY 123 L (Y)(R)(H)(T) L(2)GSWQ 221 A (Y)(R)(KE)(H) 215 V S(1)HW(68)V(3) 0.20 124 P (R)(Y)(H)(T) Y(6)F(13)NG(1) 180 T (R)(K)(H)(Q) A(1)TRILMPKCQX 139 M (Y)(T)(H)(R) 227 I V(73)I(13).F(1) 0.20 SL(6)TA(2)PGHMQ Table 11. Disruptive mutations for the surface patch in 2aipA. 123 L L(87)M(1)GEF(1) 0.21 I(2)RDV(3)QWNAP TCSK 221 A A(60)N(3)EG(28) 0.22 3 NOTES ON USING TRACE RESULTS continued in next column 3.1 Coverage Trace results are commonly expressed in terms of coverage: the residue is important if its “coverage” is small - that is if it belongs to some small top percentage of residues [100% is all of the residues

6 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 COVERAGE couple of residues should affect the protein somehow, with the exact

effects to be determined experimentally. V 3.2 Known substitutions 100% 50% 30% 5% One of the table columns is “substitutions” - other amino acid types 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 to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has V an R at that position, it is advisable to try anything, but RVK. Conver- RELATIVE IMPORTANCE 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 Fig. 10. Coloring scheme used to color residues by their relative importance. 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 guide - due to rounding errors these percentages often do not add up [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to 100%. tively [KHR], or negatively [DE] charged, aromatic [WFYH], long aliphatic chain [EKRQM], OH-group possession [SDETY ], 3.3 Surface and NH2 group possession [NQRK]. The suggestions are listed 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

7 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 file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ 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 seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.2 CE To map ligand binding sites from different of service for comparative analysis of proteins.” Bioinformatics source structures, report maker uses the CE program: 22:1656-7. http://cl.sdsc.edu/ . Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ranking visualization program was written by Ivica Res.ˇ report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- is copyrighted by Lichtarge Lab, Baylor College of Medicine, 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , Houston. which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 4.7 Attachments Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following files should accompany this report: by [email protected] November 18,2002, • 2aipA.complex.pdb - coordinates of 2aipA with all of its inter- http://www.cmbi.kun.nl/gv/dssp/descrip.html. acting partners 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • 2aipA.etvx - ET viewer input file for 2aipA gnment as a starting point for the analysis (sequences shorter than • 2aipA.cluster report.summary - Cluster report summary for 75% of the query are taken out, however); R. Schneider, A. de 2aipA Daruvar, and C. Sander. ”The HSSP database of protein structure- • 2aipA.ranks - Ranks file in sequence order for 2aipA sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 2aipA.clusters - Cluster descriptions for 2aipA http://swift.cmbi.kun.nl/swift/hssp/ • 2aipA.msf - the multiple sequence alignment used for the chain 4.3.5 LaTex The text for this report was processed using LATEX; 2aipA Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 2aipA.descr - description of sequences used in 2aipA msf Wesley,” Reading, Mass. (1986). • 2aipA.ranks sorted - full listing of residues and their ranking for 4.3.6 Muscle When making alignments “from scratch”, report 2aipA maker uses Muscle alignment program: Edgar, Robert C. (2004), • 2aipA.2aipSO4303.if.pml - Pymol script for Figure 5 ”MUSCLE: multiple sequence alignment with high accuracy and • 2aipA.cbcvg - used by other 2aipA – related pymol scripts high throughput.” Nucleic Acids Research 32(5), 1792-97. • 2aipA.2aipSO4301.if.pml - Pymol script for Figure 6 http://www.drive5.com/muscle/ • 2aipA.2aipGOL401.if.pml - Pymol script for Figure 7 4.3.7 Pymol The figures in this report were produced using • 2aipA.2aipANAG601.if.pml - Pymol script for Figure 8 Pymol. The scripts can be found in the attachment. Pymol