Pages 1–8 1hjs Evolutionary trace report by report maker April 15, 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 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 1hjs): Title: Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and ph optimum. Compound: Mol id: 1; molecule: beta-1,4-galactanase; chain: a, b, c, d; ec: 3.2.1.89; engineered: yes; other details: 2-n-acetyl-beta-d- glucose(residue 601) linked to asn 111 in the four molecules Organism, scientific name: Thielavia Heterothallica; 1hjs contains a single unique chain 1hjsA (332 residues long) and CONTENTS its homologues 1hjsD, 1hjsC, and 1hjsB.

1 Introduction 1 2 CHAIN 1HJSA 2.1 P83692 overview 2 Chain 1hjsA 1 2.1 P83692 overview 1 From SwissProt, id P83692, 96% identical to 1hjsA: 2.2 Multiple sequence alignment for 1hjsA 1 Description: Arabinogalactan endo-1,4-beta-galactosidase (EC 2.3 Residue ranking in 1hjsA 1 3.2.1.89) (Endo-1,4- beta-galactanase) (Galactanase). 2.4 Top ranking residues in 1hjsA and their position on Organism, scientific name: Thielavia heterothallica (Mycelio- the structure 2 phthora thermophila). 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Eukaryota; Fungi; ; ; 2.4.2 Overlap with known functional surfaces at ; Sordariomycetidae; ; ; 25% coverage. 2 Corynascus. 2.4.3 Possible novel functional surfaces at 25% Catalytic activity: Endohydrolysis of 1,4-beta-D-galactosidic linka- coverage. 4 ges in arabinogalactans. Miscellaneous: Has a pH range of 5.5-8.5 with optimum of 7.0; and 3 Notes on using trace results 6 a temperature optimum of 65 degrees Celsius at pH 6.5. 3.1 Coverage 6 Similarity: Belongs to the glycosyl hydrolase 53 family. 3.2 Known substitutions 6 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 6 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 6 EMBL outstation - the European Bioinformatics Institute. There are 3.5 Annotation 7 no restrictions on its use as long as its content is in no way modified 3.6 Mutation suggestions 7 and this statement is not removed.

4 Appendix 7 2.2 Multiple sequence alignment for 1hjsA 4.1 File formats 7 For the chain 1hjsA, the alignment 1hjsA.msf (attached) with 93 4.2 Color schemes used 7 sequences was used. The alignment was downloaded from the HSSP 4.3 Credits 7 database, and fragments shorter than 75% of the query as well as

1 Lichtarge lab 2006 2.4 Top ranking residues in 1hjsA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1hjsA colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 1-166 in 1hjsA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

Fig. 2. Residues 167-332 in 1hjsA colored by their relative importance. (See Appendix, Fig.10, for the coloring scheme.)

duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1hjsA.msf. Its statistics, from the Fig. 3. Residues in 1hjsA, colored by their relative importance. Clockwise: alistat program are the following: front, back, top and bottom views.

Format: MSF Number of sequences: 93 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 28259 top 25% of all residues, this time colored according to clusters they Smallest: 92 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 332 in Table 1. Average length: 303.9 Alignment length: 332 Table 1. Average identity: 36% cluster size member Most related pair: 99% color residues Most unrelated pair: 0% red 70 6,8,10,11,45,47,49,50,52,55 Most distant seq: 38% 58,77,78,79,80,81,82,83,84 86,87,88,89,92,95,97,98,105 112,113,127,130,131,132,133 Furthermore, <1% of residues show as conserved in this ali- 134,135,139,141,156,160,163 gnment. 164,165,175,178,179,180,182 The alignment consists of 4% eukaryotic ( 4% fungi), and 17% 192,206,207,209,210,212,214 prokaryotic sequences. (Descriptions of some sequences were not 215,217,239,245,248,272,273 readily available.) The file containing the sequence descriptions can 294,295,296,297,298,300,307 be found in the attachment, under the name 1hjsA.descr. blue 6 16,19,66,68,69,73 yellow 3 36,40,42 2.3 Residue ranking in 1hjsA green 2 290,292 purple 2 120,124 The 1hjsA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 1hjsA can be found in the file called 1hjsA.ranks sorted in the attachment.

2 type in the bracket; noc/bb: number of contacts with the ligand, with the num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations 73 G (R)(FKWH)(E)(M) 42 N (FYWH)(R)(TVA)(CG)

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

Fig. 4. Residues in 1hjsA, 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

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

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. Fig. 5. Residues in 1hjsA, at the interface with PEG, colored by their relative A˚ PEG binding site. By analogy with 1hjsB – 1hjsBPEG801 inter- importance. The ligand (PEG) is colored green. Atoms further than 30 away from the geometric center of the ligand, as well as on the line of sight to the face. Table 2 lists the top 25% of residues at the interface with ligand were removed. (See Appendix for the coloring scheme for the protein 1hjsBPEG801 (peg). The following table (Table 3) suggests possible chain 1hjsA.) disruptive replacements for these residues (see Section 3.6).

Table 2. Figure 5 shows residues in 1hjsA colored by their importance, at the res type subst’s cvg noc/ dist antn interface with 1hjsBPEG801. (%) bb (A˚ ) Sulfate ion binding site. Table 4 lists the top 25% of residues 73 G G(87) 0.10 8/8 3.04 at the interface with 1hjsASO4701 (sulfate ion). The following table .(3) (Table 5) suggests possible disruptive replacements for these residues N(7) (see Section 3.6). D(2) Table 4. 42 N D(10) 0.14 17/7 3.58 site res type subst’s cvg noc/ dist antn N(84) (%) bb (A˚ ) .(3) 98 W W(97) 0.01 12/0 3.02 site T(1) .(2) 50 V V(58) 0.16 4/0 3.74 Table 2. The top 25% of residues in 1hjsA at the interface with continued in next column PEG.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each

3 Table 4. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) N(27) .(2) H(7) G(1) I(1) T(1) Q(1) 112 Y H(22) 0.17 6/1 3.44 Y(63) .(3) F(9) S(1)

Table 4. The top 25% of residues in 1hjsA 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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Fig. 6. Residues in 1hjsA, at the interface with sulfate ion, colored by their relative importance. The ligand (sulfate ion) is colored green. Atoms further Table 5. than 30A˚ away from the geometric center of the ligand, as well as on the line res type disruptive of sight to the ligand were removed. (See Appendix for the coloring scheme mutations for the protein chain 1hjsA.) 98 W (KE)(TQD)(SNCG)(R) 50 V (YER)(K)(H)(D) 112 Y (K)(Q)(M)(E) Table 7. res type disruptive mutations Table 5. List of disruptive mutations for the top 25% of residues in 1hjsA, that are at the interface with sulfate ion. 42 N (FYWH)(R)(TVA)(CG)

Table 7. List of disruptive mutations for the top 25% of residues in 1hjsA, Figure 6 shows residues in 1hjsA colored by their importance, at the that are at the interface with sulfate ion. interface with 1hjsASO4701. Sulfate ion binding site. Table 6 lists the top 25% of residues at the interface with 1hjsASO4703 (sulfate ion). The following table Figure 7 shows residues in 1hjsA colored by their importance, at the (Table 7) suggests possible disruptive replacements for these residues interface with 1hjsASO4703. (see Section 3.6). 2.4.3 Possible novel functional surfaces at 25% coverage. One Table 6. group of residues is conserved on the 1hjsA surface, away from (or res type subst’s cvg noc/ dist antn susbtantially larger than) other functional sites and interfaces reco- (%) bb (A˚ ) gnizable in PDB entry 1hjs. It is shown in Fig. 8. The right panel 42 N D(10) 0.14 12/0 3.34 site shows (in blue) the rest of the larger cluster this surface belongs to. N(84) The residues belonging to this surface ”patch” are listed in Table .(3) 8, while Table 9 suggests possible disruptive replacements for these T(1) residues (see Section 3.6). Table 8. Table 6. The top 25% of residues in 1hjsA at the interface with sulfate res type substitutions(%) cvg antn ion.(Field names: res: residue number in the PDB entry; type: amino acid 81 H H(96).(2)L(1) 0.00 type; substs: substitutions seen in the alignment; with the percentage of each 84 D D(96).(2)N(1) 0.01 type in the bracket; noc/bb: number of contacts with the ligand, with the num- 95 P P(97).(2) 0.01 ber of contacts realized through backbone atoms given in the bracket; dist: 98 W W(97).(2) 0.01 site distance of closest apporach to the ligand. ) 134 N N(96).(2)S(1) 0.02 135 E E(97).(2) 0.02 continued in next column

4 Table 8. continued res type substitutions(%) cvg antn .(5) 40 G G(93).(3)D(1) 0.07 E(1)Q(1) 47 R R(88).(3)K(8) 0.08 296 W W(91).(7)G(1) 0.08 19 G G(91).(3)P(1) 0.09 N(4) 16 E E(84)K(5).(3) 0.10 A(3)N(1)D(1) V(1) 73 G G(87).(3)N(7) 0.10 D(2) 52 P P(88).(3)A(5) 0.11 F(1)Y(1)N(1) 87 A A(83).(3)T(9) 0.12 V(3) 68 R R(83)Y(1).(3) 0.13 A(1)Q(1)K(3) W(1)E(3)D(1) N(1) Fig. 7. Residues in 1hjsA, at the interface with sulfate ion, colored by their 42 N D(10)N(84).(3) 0.14 site relative importance. The ligand (sulfate ion) is colored green. Atoms further T(1) than 30A˚ away from the geometric center of the ligand, as well as on the line 307 G G(79).(16)P(2) 0.14 of sight to the ligand were removed. (See Appendix for the coloring scheme A(1)R(1) for the protein chain 1hjsA.) 36 L L(72).(3)M(6) 0.15 F(13)R(1)V(3) 50 V V(58)N(27).(2) 0.16 H(7)G(1)I(1) T(1)Q(1) 112 Y H(22)Y(63).(3) 0.17 F(9)S(1) 298 P P(81).(7)A(3) 0.17 G(4)T(1)S(2) 55 G G(73).(3)H(3) 0.18 K(1)N(1)T(8) S(4)P(1)D(1) Q(1)W(1)E(1) Fig. 8. A possible active surface on the chain 1hjsA. The larger cluster it 217 Y N(1)W(54)Y(25) 0.19 belongs to is shown in blue. V(4)F(1)I(1) .(3)K(1)A(3) D(2)Q(1)S(1) Table 8. continued 22 Y F(38)Y(51).(3) 0.20 res type substitutions(%) cvg antn W(5)L(1) 86 W W(91).(3)Y(1) 0.03 97 G A(68).(2)G(18) 0.20 F(4) S(5)E(1)V(2) 214 Y Y(96).(3) 0.03 Q(2) 49 W W(89).(2)A(1) 0.04 300 W A(5)W(66).(12) 0.20 F(7) C(5)F(1)S(5) 212 S S(94)K(1).(3) 0.04 G(1)E(1)V(1) L(1) 180 D A(35)D(38)G(1) 0.21 88 D D(93).(3)S(1) 0.05 E(6)T(6).(2) E(1)A(1) S(4)H(3)N(2) 89 P P(92).(3)K(1) 0.05 continued in next column G(3) 245 E E(92)D(1)K(1) 0.06 continued in next column

5 Table 8. continued res type substitutions(%) cvg antn 58 N S(11)D(41).(3) 0.25 N(35)G(4)A(1) Q(1)W(1)

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

Table 9. res type disruptive mutations 81 H (E)(T)(D)(Q) Fig. 9. Another possible active surface on the chain 1hjsA. The larger cluster it belongs to is shown in blue. 84 D (R)(FWH)(YVCAG)(T) 95 P (YR)(TH)(SCG)(KE) 98 W (KE)(TQD)(SNCG)(R) Table 10. 134 N (Y)(FWH)(TR)(EVCAG) res type substitutions(%) cvg 135 E (FWH)(VCAG)(YR)(T) 207 D D(94).(3)E(1) 0.08 86 W (K)(E)(Q)(D) N(1) 214 Y (K)(QM)(NVLAPI)(ER) 165 K K(60)R(30).(2) 0.22 49 W (KE)(QD)(T)(NR) A(2)D(4)L(1) 212 S (R)(FWH)(KY)(QM) 239 K S(5)K(74)C(3) 0.22 88 D (R)(H)(FW)(K) R(2)Q(2)M(1) 89 P (Y)(R)(H)(T) .(5)V(2)A(3) 245 E (FW)(H)(YVCAG)(R) L(1) 40 G (R)(FWH)(K)(Y) 164 I V(67).(2)I(18) 0.24 47 R (T)(D)(Y)(VCAG) T(5)L(4)A(1) 296 W (KE)(D)(Q)(TNR) F(1) 19 G (R)(E)(KH)(FW) 175 I A(1)V(45).(3) 0.25 16 E (H)(FW)(Y)(R) I(43)T(4)S(1) 73 G (R)(FKWH)(E)(M) L(1)Y(1) 52 P (R)(Y)(T)(KEH) 87 A (KR)(E)(Y)(QH) Table 10. Residues forming surface ”patch” in 1hjsA. 68 R (T)(YD)(CG)(VA) 42 N (FYWH)(R)(TVA)(CG) 307 G (E)(KR)(D)(H) Table 11. 36 L (Y)(T)(R)(H) 50 V (YER)(K)(H)(D) res type disruptive 112 Y (K)(Q)(M)(E) mutations 298 P (R)(Y)(H)(K) 207 D (R)(FWH)(YVCAG)(TK) 55 G (R)(K)(E)(FWH) 165 K (Y)(T)(FW)(CG) 217 Y (K)(QR)(M)(E) 239 K (Y)(FW)(T)(H) 22 Y (K)(Q)(E)(MR) 164 I (R)(Y)(H)(K) 97 G (R)(K)(H)(E) 175 I (R)(Y)(H)(K) 300 W (K)(E)(Q)(R) 180 D (R)(FWH)(K)(Y) Table 11. Disruptive mutations for the surface patch in 1hjsA. 58 N (Y)(H)(FW)(R)

Table 9. Disruptive mutations for the surface patch in 1hjsA. 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Another group of surface residues is shown in Fig.9. The right panel Trace results are commonly expressed in terms of coverage: the resi- shows (in blue) the rest of the larger cluster this surface belongs to. due is important if its “coverage” is small - that is if it belongs to The residues belonging to this surface ”patch” are listed in Table 10, some small top percentage of residues [100% is all of the residues while Table 11 suggests possible disruptive replacements for these in a chain], according to trace. The ET results are presented in the residues (see Section 3.6). 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, is an open-source application copyrighted by DeLano Scien- tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. tific LLC (2005). For more information about Pymol see The colors used to distinguish the residues by the estimated http://pymol.sourceforge.net/. (Note for Windows evolutionary pressure they experience can be seen in Fig. 10. users: the attached package needs to be unzipped for Pymol to read the scripts and launch the viewer.) 4.3 Credits 4.4 Note about ET Viewer 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- Dan Morgan from the Lichtarge lab has developed a visualization stics include the format, the number of sequences, the total number tool specifically for viewing trace results. If you are interested, please of residues, the average and range of the sequence lengths, and the visit: alignment length (e.g. including gap characters). Also shown are http://mammoth.bcm.tmc.edu/traceview/ some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of The viewer is self-unpacking and self-installing. Input files to be used exact identities and len1, len2 are the unaligned lengths of the two with ETV (extension .etvx) can be found in the attachment to the sequences. The ”average percent identity”, ”most related pair”, and main report. ”most unrelated pair” of the alignment are the average, maximum, 4.5 Citing this work and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best The method used to rank residues and make predictions in this report relative) for all N sequences, then finding the minimum of these N can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of numbers (hence, the most outlying sequence). alistat is copyrighted Evolution-Entropy Hybrid Methods for Ranking of Protein Residues by HHMI/Washington University School of Medicine, 1992-2001, by Importance” J. Mol. Bio. 336: 1265-82. For the original version and freely distributed under the GNU General Public License. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- tionary Trace Method Defines Binding Surfaces Common to Protein 4.3.2 CE To map ligand binding sites from different Families” J. Mol. Bio. 257: 342-358. source structures, report maker uses the CE program: report maker itself is described in Mihalek I., I. Res and O. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type ”Protein structure alignment by incremental combinatorial extension of service for comparative analysis of proteins.” Bioinformatics (CE) of the optimal path . Protein Engineering 11(9) 739-747. 22:1656-7. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4.6 About report maker 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , report maker was written in 2006 by Ivana Mihalek. The 1D ran- which is roughly the area needed for one water molecule to come in king visualization program was written by Ivica Res.ˇ report maker the contact with the residue. DSSP is copyrighted by W. Kabsch, C. is copyrighted by Lichtarge Lab, Baylor College of Medicine, Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version Houston. 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- • 1hjsA.complex.pdb - coordinates of 1hjsA with all of its inter- gnment as a starting point for the analysis (sequences shorter than acting partners 75% of the query are taken out, however); R. Schneider, A. de • 1hjsA.etvx - ET viewer input file for 1hjsA Daruvar, and C. Sander. ”The HSSP database of protein structure- • 1hjsA.cluster report.summary - Cluster report summary for sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1hjsA http://swift.cmbi.kun.nl/swift/hssp/ • 1hjsA.ranks - Ranks file in sequence order for 1hjsA • 1hjsA.clusters - Cluster descriptions for 1hjsA 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 1hjsA.msf - the multiple sequence alignment used for the chain Wesley,” Reading, Mass. (1986). 1hjsA • 1hjsA.descr - description of sequences used in 1hjsA msf 4.3.6 Muscle When making alignments “from scratch”, report • maker uses Muscle alignment program: Edgar, Robert C. (2004), 1hjsA.ranks sorted - full listing of residues and their ranking for ”MUSCLE: multiple sequence alignment with high accuracy and 1hjsA high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1hjsA.1hjsBPEG801.if.pml - Pymol script for Figure 5 • 1hjsA.cbcvg - used by other 1hjsA – related pymol scripts http://www.drive5.com/muscle/ • 1hjsA.1hjsASO4701.if.pml - Pymol script for Figure 6 4.3.7 Pymol The figures in this report were produced using • 1hjsA.1hjsASO4703.if.pml - Pymol script for Figure 7 Pymol. The scripts can be found in the attachment. Pymol

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