Pages 1–8 2d3p Evolutionary trace report by report maker June 6, 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 2d3p): Title: Cratylia floribunda seed lectin crystallized at basic ph Compound: Mol id: 1; molecule: lectin alpha chain; chain: a, b, c, d Organism, scientific name: Cratylia Floribunda; 2d3p contains a single unique chain 2d3pA (236 residues long) and its homologues 2d3pD, 2d3pC, and 2d3pB.

CONTENTS 2 CHAIN 2D3PA 2.1 P81517 overview 1 Introduction 1 From SwissProt, id P81517, 99% identical to 2d3pA: 2 Chain 2d3pA 1 Description: Lectin alpha chain [Contains: Lectin beta chain; Lectin 2.1 P81517 overview 1 gamma chain]. 2.2 Multiple sequence alignment for 2d3pA 1 Organism, scientific name: Cratylia floribunda. 2.3 Residue ranking in 2d3pA 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.4 Top ranking residues in 2d3pA and their position on Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core the structure 2 eudicotyledons; ; eurosids I; ; ; Papilionoi- 2.4.1 Clustering of residues at 25% coverage. 2 deae; ; Cratylia. 2.4.2 Overlap with known functional surfaces at Function: D-mannose/D-glucose-binding lectin. Mixture of 60lectin 25% coverage. 2 and 40 2.4.3 Possible novel functional surfaces at 25% Subunit: pH-dependent homodimer of alpha chains at pH values coverage. 5 lower than 6.5 and a homotetramer of alpha chains above this value. Tissue specificity: Seed. 3 Notes on using trace results 6 Ptm: The beta and gamma chains are produced by partial proteolytic 3.1 Coverage 6 processing of the lectin alpha chain by an asparaginyl endopeptidase. 3.2 Known substitutions 6 Mass spectrometry: MW=25397; MW ERR=3; 3.3 Surface 6 METHOD=Electrospray; RANGE=1-236; NOTE=Ref.2. 3.4 Number of contacts 7 Mass spectrometry: MW=12847; MW ERR=2; 3.5 Annotation 7 METHOD=Electrospray; RANGE=1-118; NOTE=Ref.2. 3.6 Mutation suggestions 7 Mass spectrometry: MW=12568; MW ERR=2; METHOD=Electrospray; RANGE=119-236; NOTE=Ref.2. 4 Appendix 7 Miscellaneous: Binds one manganese (or other transition metal) ion 4.1 File formats 7 and one calcium ion. The metal ions are essential for the saccharide- 4.2 Color schemes used 7 binding and cell-agglutinating activities. 4.3 Credits 7 Similarity: Belongs to the leguminous lectin family.

1 Lichtarge lab 2006 in 2d3pA can be found in the file called 2d3pA.ranks sorted in the attachment. 2.4 Top ranking residues in 2d3pA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 2d3pA colored by their importance: bright red and yellow indicate more conser- Fig. 1. Residues 1-118 in 2d3pA colored by their relative importance. (See ved/important residues (see Appendix for the coloring scheme). A Appendix, Fig.9, for the coloring scheme.) Pymol script for producing this figure can be found in the attachment.

Fig. 2. Residues 119-236 in 2d3pA colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

About: This Swiss-Prot entry is copyright. It is produced through a collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are 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 2d3pA For the chain 2d3pA, the alignment 2d3pA.msf (attached) with 26 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 2d3pA.msf. Its statistics, from the Fig. 3. Residues in 2d3pA, colored by their relative importance. Clockwise: alistat program are the following: front, back, top and bottom views.

Format: MSF Number of sequences: 26 Total number of residues: 5760 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 188 top 25% of all residues, this time colored according to clusters they Largest: 236 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 221.5 in Table 1. Alignment length: 236 Average identity: 36% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 19% color residues Most distant seq: 31% red 51 5,6,7,8,9,10,14,19,20,24,26 27,28,29,31,34,37,52,54,56 60,61,62,63,75,76,77,79,80 Furthermore, 2% of residues show as conserved in this alignment. 81,85,86,87,89,92,93,94,95 The alignment consists of 34% eukaryotic ( 34% plantae) 97,98,102,104,106,108,109 sequences. (Descriptions of some sequences were not readily availa- 111,113,171,178,220,231 ble.) The file containing the sequence descriptions can be found in blue 3 48,49,50 the attachment, under the name 2d3pA.descr. yellow 2 223,233 2.3 Residue ranking in 2d3pA Table 1. Clusters of top ranking residues in 2d3pA. The 2d3pA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues

2 Fig. 4. Residues in 2d3pA, 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.

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. Calcium ion binding site. Table 2 lists the top 25% of residues at the interface with 2d3pCA237 (calcium ion). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 10 D D(100) 0.03 4/0 2.41 24 H H(100) 0.03 1/0 4.74 14 N N(92) 0.07 6/2 2.37 D(7) 19 D D(80) 0.15 4/0 2.38 E(7) N(7) Y(3) 12 Y F(7) 0.25 5/4 2.39 Y(76) R(7) H(7)

Table 2. The top 25% of residues in 2d3pA at the interface with calcium 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. )

3 Table 3. Table 4. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 10 D (R)(FWH)(KYVCAG)(TQM) K(3) 24 H (E)(TQMD)(SNKVCLAPIG)(YR) 108 S S(84) 0.13 11/3 2.71 14 N (Y)(FWH)(TR)(VCAG) G(3) 19 D (R)(FWH)(VA)(KCG) N(3) 12 Y (K)(EQM)(NVLAPDI)(R) Y(7) 75 V L(57) 0.14 1/1 4.80 Table 3. List of disruptive mutations for the top 25% of residues in V(38) 2d3pA, that are at the interface with calcium ion. Y(3) 62 S S(76) 0.16 19/2 3.84 .(7) T(7) K(3) A(3) 63 A V(53) 0.18 4/4 4.11 A(42) M(3) 49 T H(26) 0.20 27/0 3.17 D(7) T(53) N(11) 76 S S(69) 0.24 1/0 4.99 Y(19) I(3) N(3) W(3)

Table 4. The top 25% of residues in 2d3pA at the interface with 2d3pD. (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. 5. Residues in 2d3pA, at the interface with calcium ion, colored by their relative importance. The ligand (calcium ion) is colored green. Atoms further Table 5. 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 res type disruptive for the protein chain 2d3pA.) mutations 109 W (KE)(QD)(TR)(N) 60 R (T)(Y)(D)(SCG) Figure 5 shows residues in 2d3pA colored by their importance, at the 108 S (KR)(M)(FQWH)(E) interface with 2d3pCA237. 75 V (K)(R)(E)(YQ) Interface with 2d3pD.Table 4 lists the top 25% of residues at the 62 S (R)(K)(FWH)(QM) interface with 2d3pD. The following table (Table 5) suggests possible 63 A (Y)(R)(KE)(H) disruptive replacements for these residues (see Section 3.6). 49 T (R)(K)(FMW)(QH) 76 S (KR)(Q)(EMH)(FW) Table 4. res type subst’s cvg noc/ dist (%) bb (A˚ ) Table 5. List of disruptive mutations for the top 25% of residues in 2d3pA, that are at the interface with 2d3pD. 109 W W(96) 0.07 6/6 3.93 A(3) 60 R N(46) 0.13 49/1 2.15 Figure 6 shows residues in 2d3pA colored by their importance, at the R(30) interface with 2d3pD. I(19) Manganese (ii) ion binding site. Table 6 lists the top 25% of resi- continued in next column dues at the interface with 2d3pMN238 (manganese (ii) ion). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6).

4 Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) 10 D D(100) 0.03 4/0 2.24 24 H H(100) 0.03 5/0 2.24 8 E E(96) 0.05 4/0 1.99 T(3) 34 S S(96) 0.06 1/0 3.92 X(3) 20 P P(84) 0.11 1/0 4.81 K(7) A(7) 19 D D(80) 0.15 5/1 1.89 E(7) N(7) Y(3)

Table 6. The top 25% of residues in 2d3pA at the interface with manga- nese (ii) 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; Fig. 6. Residues in 2d3pA, at the interface with 2d3pD, colored by their rela- dist: distance of closest apporach to the ligand. ) tive importance. 2d3pD is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 2d3pA.) Table 7. res type disruptive mutations 10 D (R)(FWH)(KYVCAG)(TQM) 24 H (E)(TQMD)(SNKVCLAPIG)(YR) 8 E (FWH)(R)(YVA)(KCG) 34 S (KR)(FQMWH)(YE)(NLPI) 20 P (Y)(R)(TH)(E) 19 D (R)(FWH)(VA)(KCG)

Table 7. List of disruptive mutations for the top 25% of residues in 2d3pA, that are at the interface with manganese (ii) ion.

Figure 7 shows residues in 2d3pA colored by their importance, at the interface with 2d3pMN238. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2d3pA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2d3p. It is shown in Fig. 8. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 8, while Table 9 suggests possible disruptive replacements for these residues (see Section 3.6). Table 8. res type substitutions(%) cvg 10 D D(100) 0.03 54 Y Y(100) 0.03 61 L L(100) 0.03 79 V V(92)I(7) 0.03 85 L L(100) 0.03 continued in next column

5 Table 8. continued res type substitutions(%) cvg P(3) 60 R N(46)R(30)I(19) 0.13 K(3) 75 V L(57)V(38)Y(3) 0.14 19 D D(80)E(7)N(7) 0.15 Y(3) 77 Y Y(80)A(7)V(3) 0.15 L(3)T(3) 62 S S(76).(7)T(7) 0.16 K(3)A(3) 63 A V(53)A(42)M(3) 0.18 56 S S(76)A(11)N(7) 0.19 G(3) 178 V R(3)V(73)I(11) 0.22 M(3)L(3)F(3) 5 V I(15)V(76)F(3) 0.23 .(3) 29 I I(73)X(3)V(15) 0.23 N(3)L(3) 37 T T(69)N(15)K(3) 0.23 Fig. 7. Residues in 2d3pA, at the interface with manganese (ii) ion, colored A(7)S(3) by their relative importance. The ligand (manganese (ii) ion) is colored green. 76 S S(69)Y(19)I(3) 0.24 Atoms further than 30A˚ away from the geometric center of the ligand, as well N(3)W(3) as on the line of sight to the ligand were removed. (See Appendix for the 11 T T(65)I(3)S(30) 0.25 coloring scheme for the protein chain 2d3pA.) 12 Y F(7)Y(76)R(7) 0.25 H(7)

Table 8. Residues forming surface ”patch” in 2d3pA.

Table 9. res type disruptive mutations 10 D (R)(FWH)(KYVCAG)(TQM) 54 Y (K)(QM)(NEVLAPIR)(D) 61 L (YR)(TH)(SKECG)(FQWD) 79 V (YR)(KE)(H)(QD) Fig. 8. A possible active surface on the chain 2d3pA. The larger cluster it 85 L (YR)(TH)(SKECG)(FQWD) belongs to is shown in blue. 86 P (Y)(T)(HR)(SCG) 31 S (KR)(FQMWH)(E)(NYLPI) 81 L (Y)(R)(TH)(SCG) Table 8. continued 7 V (YR)(KE)(H)(QD) res type substitutions(%) cvg 34 S (KR)(FQMWH)(YE)(NLPI) 86 P P(96)K(3) 0.03 14 N (Y)(FWH)(TR)(VCAG) 31 S S(96)G(3) 0.04 80 D (R)(FWH)(Y)(VCAG) 81 L L(92)M(7) 0.04 87 E (FWH)(R)(YVCAG)(T) 7 V V(96)L(3) 0.05 52 I (YR)(H)(TKE)(SQCDG) 34 S S(96)X(3) 0.06 6 A (KR)(E)(YH)(Q) 14 N N(92)D(7) 0.07 20 P (Y)(R)(TH)(E) 80 D D(92)N(7) 0.08 113 S (R)(K)(H)(FQW) 87 E E(88)D(11) 0.09 60 R (T)(Y)(D)(SCG) 52 I I(73)V(26) 0.10 75 V (K)(R)(E)(YQ) 6 A A(92)T(3).(3) 0.11 19 D (R)(FWH)(VA)(KCG) 20 P P(84)K(7)A(7) 0.11 continued in next column 113 S S(88)G(3)T(3) 0.11 continued in next column

6 Table 9. continued should not be automatically dropped from consideration when choo- res type disruptive sing the set for mutagenesis. (Especially if they form a cluster with mutations the surface residues.) 77 Y (K)(QR)(E)(M) 62 S (R)(K)(FWH)(QM) 3.4 Number of contacts 63 A (Y)(R)(KE)(H) Another column worth noting is denoted “noc/bb”; it tells the num- 56 S (R)(K)(H)(FW) ber of contacts heavy atoms of the residue in question make across 178 V (Y)(E)(R)(K) the interface, as well as how many of them are realized through the 5 V (KER)(Y)(QD)(H) backbone atoms (if all or most contacts are through the backbone, 29 I (Y)(R)(H)(T) mutation presumably won’t have strong impact). Two heavy atoms 37 T (R)(K)(FWH)(M) are considered to be “in contact” if their centers are closer than 5A˚ . 76 S (KR)(Q)(EMH)(FW) 11 T (R)(K)(H)(FQW) 3.5 Annotation 12 Y (K)(EQM)(NVLAPDI)(R) If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” Table 9. Disruptive mutations for the surface patch in 2d3pA. appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue).

3 NOTES ON USING TRACE RESULTS 3.6 Mutation suggestions 3.1 Coverage Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino couple of residues should affect the protein somehow, with the exact acid, and they are grouped in round brackets if they appear equally effects to be determined experimentally. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 4 APPENDIX an R at that position, it is advisable to try anything, but RVK. Conver- 4.1 File formats sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file: is given in the immediately following bracket. No percentage is given • alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • guide - due to rounding errors these percentages often do not add up residue# residue number in the PDB file to 100%. • type amino acid type • rank rank of the position according to older version of ET 3.3 Surface • variability has two subfields: To detect candidates for novel functional interfaces, first we look for 1. number of different amino acids appearing in in this column residues that are solvent accessible (according to DSSP program) by of the alignment 2 at least 10A˚ , which is roughly the area needed for one water mole- 2. their type cule to come in the contact with the residue. Furthermore, we require • that these residues form a “cluster” of residues which have neighbor rho ET score - the smaller this value, the lesser variability of within 5A˚ from any of their heavy atoms. this position across the branches of the tree (and, presumably, Note, however, that, if our picture of protein evolution is correct, the greater the importance for the protein) the neighboring residues which are not surface accessible might be • cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller

7 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. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, COVERAGE http://www.cmbi.kun.nl/gv/dssp/descrip.html.

V 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 100% 50% 30% 5% gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/ V 4.3.5 LaTex The text for this report was processed using LATEX; RELATIVE IMPORTANCE Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). Fig. 9. Coloring scheme used to color residues by their relative importance. 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 9. 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 ran- king 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, ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Houston.

8 4.7 Attachments • 2d3pA.msf - the multiple sequence alignment used for the chain The following files should accompany this report: 2d3pA • 2d3pA.descr - description of sequences used in 2d3pA msf • 2d3pA.ranks sorted - full listing of residues and their ranking • 2d3pA.complex.pdb - coordinates of 2d3pA with all of its for 2d3pA interacting partners • 2d3pA.2d3pCA237.if.pml - Pymol script for Figure 5 • 2d3pA.etvx - ET viewer input file for 2d3pA • 2d3pA.cbcvg - used by other 2d3pA – related pymol scripts • 2d3pA.cluster report.summary - Cluster report summary for • 2d3pA 2d3pA.2d3pD.if.pml - Pymol script for Figure 6 • • 2d3pA.ranks - Ranks file in sequence order for 2d3pA 2d3pA.2d3pMN238.if.pml - Pymol script for Figure 7 • 2d3pA.clusters - Cluster descriptions for 2d3pA

9