Pages 1–8 1kxr Evolutionary trace report by report maker June 29, 2010

4.3.1 Alistat 7 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 7 4.4 Note about ET Viewer 8 4.5 Citing this work 8 4.6 About report maker 8 4.7 Attachments 8

1 INTRODUCTION From the original Data Bank entry (PDB id 1kxr): Title: Crystal structure of calcium-bound core of i Compound: Mol id: 1; molecule: thiol protease domains i and ii; chain: a, b; fragment: residues 27-356; synonym: calpain i protease core; ec: 3.4.22.17; engineered: yes; mutation: yes Organism, scientific name: Rattus Norvegicus; 1kxr contains a single unique chain 1kxrB (321 residues long) and its homologue 1kxrA. CONTENTS

1 Introduction 1 2 CHAIN 1KXRB 2.1 P97571 overview 2 Chain 1kxrB 1 2.1 P97571 overview 1 From SwissProt, id P97571, 99% identical to 1kxrB: 2.2 Multiple sequence alignment for 1kxrB 1 Description: Calpain-1 catalytic subunit (EC 3.4.22.52) (Calpain- 2.3 Residue ranking in 1kxrB 1 1 large subunit) (Calcium-activated neutral proteinase 1) (CANP 1) 2.4 Top ranking residues in 1kxrB and their position on (Calpain mu-type) (muCANP) (Micromolar-calpain). the structure 2 Organism, scientific name: Rattus norvegicus (Rat). 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Verte- 2.4.2 Overlap with known functional surfaces at brata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; 25% coverage. 3 Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Rattus. 2.4.3 Possible novel functional surfaces at 25% Function: Calcium-regulated non-lysosomal thiol-protease which coverage. 4 catalyze limited proteolysis of substrates involved in cytoskeletal remodelling and signal tranduction. 3 Notes on using trace results 6 Catalytic activity: Broad endopeptidase specificity. 3.1 Coverage 6 Cofactor: Binds 3 calcium ions (By similarity). 3.2 Known substitutions 6 Enzyme regulation: Activated by micromolar concentrations of 3.3 Surface 6 calcium and inhibited by calpastatin. 3.4 Number of contacts 6 Subunit: Forms a heterodimer with a small (regulatory) subunit 3.5 Annotation 6 (CAPNS1). 3.6 Mutation suggestions 6 Subcellular location: Cytoplasmic; Translocates to the plasma membrane upon Ca(2+) binding (By similarity). 4 Appendix 7 Tissue specificity: Ubiquitous. 4.1 File formats 7 Similarity: Belongs to the peptidase C2 family. 4.2 Color schemes used 7 Similarity: Contains 1 calpain catalytic domain. 4.3 Credits 7 Similarity: Contains 4 EF-hand domains.

1 Lichtarge lab 2006 Fig. 1. Residues 33-192 in 1kxrB colored by their relative importance. (See Fig. 2. Residues 193-353 in 1kxrB colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.) Appendix, Fig.9, for the coloring scheme.)

About: This Swiss-Prot entry is copyright. It is produced through a importance: bright red and yellow indicate more conserved/important collaboration between the Swiss Institute of Bioinformatics and the residues (see Appendix for the coloring scheme). A Pymol script for EMBL outstation - the European Bioinformatics Institute. There are producing this figure can be found in the attachment. 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 1kxrB For the chain 1kxrB, the alignment 1kxrB.msf (attached) with 298 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 1kxrB.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 298 Total number of residues: 89255 Smallest: 137 Largest: 321 Average length: 299.5 Alignment length: 321 Average identity: 37% Most related pair: 99% Most unrelated pair: 0% Most distant seq: 32%

Furthermore, <1% of residues show as conserved in this ali- Fig. 3. Residues in 1kxrB, colored by their relative importance. Clockwise: gnment. front, back, top and bottom views. The alignment consists of 45% eukaryotic ( 31% vertebrata, 4% arthropoda, 1% fungi, 1% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the sequence descriptions can be found in the attachment, under the name top 25% of all residues, this time colored according to clusters they 1kxrB.descr. belong to. The clusters in Fig.4 are composed of the residues listed 2.3 Residue ranking in 1kxrB in Table 1. The 1kxrB sequence is shown in Figs. 1–2, with each residue colored Table 1. according to its estimated importance. The full listing of residues cluster size member in 1kxrB can be found in the file called 1kxrB.ranks sorted in the color residues attachment. red 81 56,58,61,85,87,88,106,109 110,112,113,114,115,116,117 2.4 Top ranking residues in 1kxrB and their position on 119,120,122,147,149,151,154 the structure continued in next column In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1kxrB colored by their

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) .(4) S(1)IW 332 G G(87) 0.14 2/2 4.49 S(3)AT .(5)YKR C(1) 309 D D(79) 0.21 4/0 2.21 K(2) E(4) P(1)V W(1) N(2)Q .(3)GA Y(1) S(2) 302 E E(78) 0.23 4/0 2.41 I(1)H D(1) G(1) Fig. 4. Residues in 1kxrB, colored according to the cluster they belong to: S(5) red, followed by blue and yellow are the largest clusters (see Appendix for N(1) the coloring scheme). Clockwise: front, back, top and bottom views. The C(2) corresponding Pymol script is attached. .(3) V(1) K(1)P Table 1. continued T(1)A cluster size member color residues 157,159,162,165,166,168,169 Table 2. The top 25% of residues in 1kxrB at the interface with calcium ion.(Field names: res: residue number in the PDB entry; type: amino acid 177,185,186,187,188,190,191 type; substs: substitutions seen in the alignment; with the percentage of each 192,193,194,195,196,197,200 type in the bracket; noc/bb: number of contacts with the ligand, with the num- 201,202,205,207,208,212,213 ber of contacts realized through backbone atoms given in the bracket; dist: 214,215,216,217,218,219,220 distance of closest apporach to the ligand. ) 267,272,273,274,291,295,296 297,298,299,302,303,305,307 309,314,332,334,335,336,340 Table 3. 345,351,353 res type disruptive mutations Table 1. Clusters of top ranking residues in 1kxrB. 334 F (K)(E)(Q)(R) 332 G (E)(K)(R)(FWD) 309 D (R)(H)(FW)(Y) 2.4.2 Overlap with known functional surfaces at 25% coverage. 302 E (H)(FW)(R)(Y) The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Table 3. List of disruptive mutations for the top 25% of residues in 1kxrB, Calcium ion binding site. Table 2 lists the top 25% of residues that are at the interface with calcium ion. at the interface with 1kxrBCA4 (calcium ion). The following table (Table 3) suggests possible disruptive replacements for these residues Figure 5 shows residues in 1kxrB colored by their importance, at the (see Section 3.6). interface with 1kxrBCA4. Table 2. Calcium ion binding site. Table 4 lists the top 25% of residues res type subst’s cvg noc/ dist at the interface with 1kxrBCA3 (calcium ion). The following table (%) bb (A˚ ) (Table 5) suggests possible disruptive replacements for these residues 334 F F(91)L 0.08 3/2 4.47 (see Section 3.6). V(1) continued in next column

3 Table 5. res type disruptive mutations 187 W (E)(K)(TD)(Q) 185 E (H)(FW)(Y)(R) 106 D (R)(H)(FW)(Y)

Table 5. List of disruptive mutations for the top 25% of residues in 1kxrB, that are at the interface with calcium ion.

Fig. 5. Residues in 1kxrB, at the interface with calcium ion, colored by their relative importance. The ligand (calcium 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 for the protein chain 1kxrB.)

Table 4. res type subst’s cvg noc/ dist (%) bb (A˚ ) 187 W W(98) 0.00 1/0 4.52 .(1)F 185 E E(82) 0.09 4/0 2.44 Fig. 6. Residues in 1kxrB, at the interface with calcium ion, colored by their C(1)K relative importance. The ligand (calcium ion) is colored green. Atoms further Q(7) than 30A˚ away from the geometric center of the ligand, as well as on the line V(2) of sight to the ligand were removed. (See Appendix for the coloring scheme .(1)P for the protein chain 1kxrB.) S(1)GD I(1)ARL 106 D D(86)E 0.17 4/0 2.40 Figure 6 shows residues in 1kxrB colored by their importance, at the Q(3)G interface with 1kxrBCA3. S(1) 2.4.3 Possible novel functional surfaces at 25% coverage. One .(3)T group of residues is conserved on the 1kxrB surface, away from (or N(1) susbtantially larger than) other functional sites and interfaces reco- A(1) gnizable in PDB entry 1kxr. It is shown in Fig. 7. The right panel C(1)L shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table Table 4. The top 25% of residues in 1kxrB at the interface with calcium 6, while Table 7 suggests possible disruptive replacements for these ion.(Field names: res: residue number in the PDB entry; type: amino acid residues (see Section 3.6). 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- Table 6. ber of contacts realized through backbone atoms given in the bracket; dist: res type substitutions(%) cvg distance of closest apporach to the ligand. ) 197 K K(97).(1)RTG 0.01 208 G G(94).(2)A(1)PR 0.02 L(1)VQC continued in next column

4 Table 6. continued res type substitutions(%) cvg NTKSCMAEY 212 E E(67)D(16)H(5) 0.15 Q(4).(2)CW(1)TL R(1)ISV 273 A A(81)S(6)Y.(4)C 0.15 T(2)EGN(1)IP(1) LH 112 L L(77)I(2)M(2) 0.16 D(4)GV(6).(2) A(2)FSEN Fig. 7. A possible active surface on the chain 1kxrB. The larger cluster it 106 D D(86)EQ(3)GS(1) 0.17 belongs to is shown in blue. .(3)TN(1)A(1) C(1)L Table 6. continued 154 W W(61)Y(6)N(9) 0.17 res type substitutions(%) cvg F(4)H(2)C(13)K 200 G G(94)QNS.(1)CIR 0.03 .(1)VRS KT 295 R LR(82)Q(2)FK(3) 0.17 202 Y Y(95)F(2).(2) 0.03 .(3)S(1)W(2) 219 G G(96)S(1).(2) 0.03 H(1)MCTA 110 G G(94)SV.(2)KRND 0.04 168 L L(82)V(1)F(4) 0.18 E I(8).(1)CQY 116 W W(85)Y(10).(2)F 0.05 220 G G(73)F(5)KY(1) 0.20 ASPN A(10)C(1).(2) 159 W W(93)F(1)LE.(1) 0.05 S(2)HT(1)DEI NARVSI 56 F .(6)F(72)Y(15) 0.21 169 P P(86)F(2)C(1) 0.05 W(4)GVAR Y(7)E.(1)SQ 201 S G(5)C(19)D(9) 0.21 113 G SG(91)N(2).(2)R 0.06 S(59)N(4).(1)A ELPQKA T(1) 115 S C(87)R(3)AS(4) 0.06 186 F F(58)T(11)L(17) 0.22 .(2)K(1)GTFYL CM(2)I(3).(1)A 87 R L(1)R(93).(3)KC 0.07 Y(2)V(1)WN TQ 177 F Y(15)F(68)GI(2) 0.24 109 Q Q(90)P(3)A(1)H 0.07 V(2).(1)CML(2) .(2)GELKR A(2)S(1)EH(1) 216 D D(84)S(2)T(5) 0.07 207 G MG(66)TA(11) 0.24 .(2)ERM(1)QIYNA Y(4)CS(10).(2)E 157 G G(91)FD(1)N(2) 0.09 LD(1)RW(1)QF .(1)TS(1)KE 215 E V(18)E(49)Q(7)L 0.25 185 E E(82)C(1)KQ(7) 0.09 A(7)T(3).(2) V(2).(1)PS(1)GD K(2)R(2)YS(1) I(1)ARL H(1)M(1)IF 218 T T(89)S(5)L(1) 0.11 353 L L(57)V(9).(26) 0.25 .(2)CV I(5)F(1)M(1) 298 W W(90)F(1)TY.(3) 0.12 HL(1)QVKR Table 6. Residues forming surface ”patch” in 1kxrB. 61 F F(90).(5)TKMHD 0.13 W(1)L 299 G G(83)S(2)LV(1) 0.14 Table 7. .(3)A(5)R(1)TDK res type disruptive I(1)CN mutations 85 W IW(82)V(8)R.(4) 0.15 197 K (Y)(FW)(T)(VA) continued in next column 208 G (E)(R)(K)(H) 200 G (E)(R)(FW)(H) continued in next column

5 Table 7. continued 8, while Table 9 suggests possible disruptive replacements for these res type disruptive residues (see Section 3.6). mutations 202 Y (K)(Q)(M)(E) Table 8. 219 G (KR)(E)(FMWH)(Q) res type substitutions(%) cvg 110 G (R)(FW)(H)(KE) 307 W W(92)Y.(3)F(1)K 0.04 116 W (K)(E)(Q)(D) LSGT 159 W (KE)(T)(D)(Q) 305 G G(92)LR(1)Q.(3) 0.09 169 P (R)(Y)(H)(TK) K(1)AEP 113 G (R)(H)(E)(FW) 335 W W(87)F(3).(4)Q 0.11 115 S (K)(R)(Q)(H) V(2)Y(1)I 87 R (D)(TY)(E)(VA) 267 N G(72)K(15)IE 0.19 109 Q (Y)(T)(FW)(H) .(6)S(1)N(1)AQ 216 D (R)(H)(FW)(Y) M(1) 157 G (R)(K)(FWH)(E) 309 D D(79)K(2)E(4) 0.21 185 E (H)(FW)(Y)(R) P(1)VW(1)N(2)Q 218 T (R)(K)(H)(Q) .(3)GAY(1)S(2) 298 W (E)(K)(D)(T) 302 E E(78)I(1)HD(1) 0.23 61 F (K)(E)(T)(Q) G(1)S(5)N(1) 299 G (R)(E)(K)(H) C(2).(3)V(1) 85 W (E)(K)(D)(TQ) K(1)PT(1)A 212 E (H)(FW)(R)(Y) 273 A (R)(K)(E)(Y) Table 8. Residues forming surface ”patch” in 1kxrB. 112 L (R)(Y)(H)(T) 106 D (R)(H)(FW)(Y) 154 W (E)(K)(D)(T) Table 9. 295 R (D)(T)(E)(Y) res type disruptive 168 L (R)(Y)(H)(T) mutations 220 G (R)(K)(E)(Q) 307 W (K)(E)(Q)(D) 56 F (E)(K)(D)(Q) 305 G (R)(EH)(Y)(FW) 201 S (R)(K)(H)(FW) 335 W (K)(E)(T)(D) 186 F (K)(E)(R)(QD) 267 N (Y)(H)(FW)(T) 177 F (K)(E)(Q)(R) 309 D (R)(H)(FW)(Y) 207 G (R)(K)(E)(H) 302 E (H)(FW)(R)(Y) 215 E (H)(FW)(Y)(R) 353 L (YR)(T)(H)(KE) Table 9. Disruptive mutations for the surface patch in 1kxrB.

Table 7. Disruptive mutations for the surface patch in 1kxrB.

Another group of surface residues is shown in Fig.8. The right panel 3 NOTES ON USING TRACE RESULTS shows (in blue) the rest of the larger cluster this surface belongs to. 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due 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 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 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 Fig. 8. Another possible active surface on the chain 1kxrB. The larger cluster seen at the same position in the alignment. These amino acid types it belongs to is shown in blue. 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 The residues belonging to this surface ”patch” are listed in Table example if the substitutions are “RVK” and the original protein has

6 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 COVERAGE 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 V to 100%. 100% 50% 30% 5%

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 at least 10A , which is roughly the area needed for one water mole- V cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor RELATIVE IMPORTANCE within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, Fig. 9. Coloring scheme used to color residues by their relative importance. the neighboring residues which are not surface accessible might be 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- • alignment# number of the position in the alignment ber of contacts heavy atoms of the residue in question make across • residue# residue number in the PDB file the interface, as well as how many of them are realized through the • type amino acid type backbone atoms (if all or most contacts are through the backbone, 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: 1. number of different amino acids appearing in in this column 3.5 Annotation of the alignment If the residue annotation is available (either from the pdb file or 2. their type from other sources), another column, with the header “annotation” • rho ET score - the smaller this value, the lesser variability of appears. Annotations carried over from PDB are the following: site this position across the branches of the tree (and, presumably, (indicating existence of related site record in PDB ), S-S (disulfide the greater the importance for the protein) bond forming residue), hb (hydrogen bond forming residue, jb (james • cvg coverage - percentage of the residues on the structure which bond forming residue), and sb (for salt bridge forming residue). have this rho or smaller • gaps percentage of gaps in this column 3.6 Mutation suggestions Mutation suggestions are completely heuristic and based on comple- 4.2 Color schemes used mentarity with the substitutions found in the alignment. Note that The following color scheme is used in figures with residues colored they are meant to be disruptive to the interaction of the protein by cluster size: black is a single-residue cluster; clusters composed of with its ligand. The attempt is made to complement the following more than one residue colored according to this hierarchy (ordered properties: small [AV GSTC], medium [LPNQDEMIK], large by descending size): red, blue, yellow, green, purple, azure, tur- [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tively [KHR], or negatively [DE] charged, aromatic [WFYH], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, long aliphatic chain [EKRQM], OH-group possession [SDETY ], DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, and NH2 group possession [NQRK]. The suggestions are listed tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. according to how different they appear to be from the original amino The colors used to distinguish the residues by the estimated acid, and they are grouped in round brackets if they appear equally evolutionary pressure they experience can be seen in Fig. 9. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 4.3 Credits disruptive) These suggestions are tentative - they might prove disrup- 4.3.1 Alistat alistat reads a multiple sequence alignment from the tive to the fold rather than to the interaction. Many researcher will file and shows a number of simple statistics about it. These stati- choose, however, the straightforward alanine mutations, especially in stics include the format, the number of sequences, the total number the beginning stages of their investigation. of residues, the average and range of the sequence lengths, and the

7 alignment length (e.g. including gap characters). Also shown are 4.4 Note about ET Viewer some percent identities. A percent pairwise alignment identity is defi- Dan Morgan from the Lichtarge lab has developed a visualization ned as (idents / MIN(len1, len2)) where idents is the number of tool specifically for viewing trace results. If you are interested, please exact identities and len1, len2 are the unaligned lengths of the two visit: sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, http://mammoth.bcm.tmc.edu/traceview/ and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant The viewer is self-unpacking and self-installing. Input files to be used seq” is calculated by finding the maximum pairwise identity (best with ETV (extension .etvx) can be found in the attachment to the relative) for all N sequences, then finding the minimum of these N main report. numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, 4.5 Citing this work and freely distributed under the GNU General Public License. The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 4.3.2 CE To map ligand binding sites from different Evolution-Entropy Hybrid Methods for Ranking of Protein Residues source structures, report maker uses the CE program: by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- ”Protein structure alignment by incremental combinatorial extension tionary Trace Method Defines Binding Surfaces Common to Protein (CE) of the optimal path . Protein Engineering 11(9) 739-747. Families” J. Mol. Bio. 257: 342-358. 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker itself is described in Mihalek I., I. Res and O. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type which is roughly the area needed for one water molecule to come in of service for comparative analysis of .” Bioinformatics the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 22:1656-7. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 4.6 About report maker by [email protected] November 18,2002, report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://www.cmbi.kun.nl/gv/dssp/descrip.html. king visualization program was written by Ivica Res.ˇ report maker 4.3.4 HSSP Whenever available, report maker uses HSSP ali- is copyrighted by Lichtarge Lab, Baylor College of Medicine, gnment as a starting point for the analysis (sequences shorter than Houston. 75% of the query are taken out, however); R. Schneider, A. de 4.7 Attachments Daruvar, and C. Sander. ”The HSSP database of protein structure- The following files should accompany this report: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • http://swift.cmbi.kun.nl/swift/hssp/ 1kxrB.complex.pdb - coordinates of 1kxrB with all of its inter- acting partners A 4.3.5 LaTex The text for this report was processed using LTEX; • 1kxrB.etvx - ET viewer input file for 1kxrB Leslie Lamport, “LaTeX: A Document Preparation System Addison- • Wesley,” Reading, Mass. (1986). 1kxrB.cluster report.summary - Cluster report summary for 1kxrB 4.3.6 Muscle When making alignments “from scratch”, report • 1kxrB.ranks - Ranks file in sequence order for 1kxrB maker uses Muscle alignment program: Edgar, Robert C. (2004), • ”MUSCLE: multiple sequence alignment with high accuracy and 1kxrB.clusters - Cluster descriptions for 1kxrB high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1kxrB.msf - the multiple sequence alignment used for the chain 1kxrB http://www.drive5.com/muscle/ • 1kxrB.descr - description of sequences used in 1kxrB msf 4.3.7 Pymol The figures in this report were produced using • 1kxrB.ranks sorted - full listing of residues and their ranking for Pymol. The scripts can be found in the attachment. Pymol 1kxrB is an open-source application copyrighted by DeLano Scien- • tific LLC (2005). For more information about Pymol see 1kxrB.1kxrBCA4.if.pml - Pymol script for Figure 5 http://pymol.sourceforge.net/. (Note for Windows • 1kxrB.cbcvg - used by other 1kxrB – related pymol scripts users: the attached package needs to be unzipped for Pymol to read • 1kxrB.1kxrBCA3.if.pml - Pymol script for Figure 6 the scripts and launch the viewer.)

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