Pages 1–9 2zbj Evolutionary trace report by report maker October 16, 2010

4.3.1 Alistat 8 4.3.2 CE 8 4.3.3 DSSP 8 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 9 4.6 About report maker 9 4.7 Attachments 9

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2zbj): Title: Crystal structure of rostrata lectin Compound: Mol id: 1; molecule: lectin alpha chain; chain: a Organism, scientific name: Dioclea Rostrata; 2zbj contains a single unique chain 2zbjA (232 residues long).

2 CHAIN 2ZBJA 2.1 P81637 overview CONTENTS From SwissProt, id P81637, 88% identical to 2zbjA: 1 Introduction 1 Description: Lectin alpha chain [Contains: Lectin beta chain; Lectin gamma-1 chain; Lectin gamma-2 chain]. 2 Chain 2zbjA 1 Organism, scientific name: Dioclea guianensis. 2.1 P81637 overview 1 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.2 Multiple sequence alignment for 2zbjA 1 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.3 Residue ranking in 2zbjA 1 eudicotyledons; ; eurosids I; ; ; Papilionoi- 2.4 Top ranking residues in 2zbjA and their position on deae; ; Dioclea. the structure 1 Function: D-mannose/D-glucose-binding lectin. Mixture of 60lectin 2.4.1 Clustering of residues at 25% coverage. 2 and 40 2.4.2 Overlap with known functional surfaces at Subunit: Equilibrium between homodimer and homotetramer. 25% coverage. 3 Ptm: The beta and gamma chains are produced by partial proteolytic 2.4.3 Possible novel functional surfaces at 25% processing of the lectin alpha chain by an asparaginyl endopeptidase. coverage. 6 Mass spectrometry: MW=25398; MW ERR=1; METHOD=Electrospray; RANGE=1-237; NOTE=Ref.1. 3 Notes on using trace results 7 Mass spectrometry: MW=12831; MW ERR=1; 3.1 Coverage 7 METHOD=Electrospray; RANGE=1-118; NOTE=Ref.1. 3.2 Known substitutions 7 Mass spectrometry: MW=12583; MW ERR=1; 3.3 Surface 7 METHOD=Electrospray; RANGE=119-237; NOTE=Ref.1. 3.4 Number of contacts 7 Mass spectrometry: MW=12012; MW ERR=1; 3.5 Annotation 7 METHOD=Electrospray; RANGE=125-237; NOTE=Ref.1. 3.6 Mutation suggestions 7 Miscellaneous: Binds one manganese (or other transition metal) ion and one calcium ion. The metal ions are essential for the saccharide- 4 Appendix 8 binding and cell-agglutinating activities. 4.1 File formats 8 Similarity: Belongs to the leguminous lectin family. 4.2 Color schemes used 8 About: This Swiss-Prot entry is copyright. It is produced through a 4.3 Credits 8 collaboration between the Swiss Institute of Bioinformatics and the

1 Lichtarge lab 2006 in 2zbjA can be found in the file called 2zbjA.ranks sorted in the attachment.

2.4 Top ranking residues in 2zbjA 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 2zbjA colored by their importance: bright red and yellow indicate more conserved/important Fig. 1. Residues 1-116 in 2zbjA colored by their relative importance. (See residues (see Appendix for the coloring scheme). A Pymol script for Appendix, Fig.11, for the coloring scheme.) producing this figure can be found in the attachment.

Fig. 2. Residues 117-237 in 2zbjA colored by their relative importance. (See Appendix, Fig.11, for the coloring scheme.)

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 2zbjA For the chain 2zbjA, the alignment 2zbjA.msf (attached) with 284 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 2zbjA.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 2zbjA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 284 Total number of residues: 62135 Smallest: 174 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 232 top 25% of all residues, this time colored according to clusters they Average length: 218.8 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 232 in Table 1. Average identity: 39% Most related pair: 99% Table 1. Most unrelated pair: 15% cluster size member Most distant seq: 33% color residues red 57 6,7,8,9,10,11,24,26,27,28,29 30,34,50,52,54,61,85,89,92 Furthermore, <1% of residues show as conserved in this ali- 93,94,97,98,109,110,111,113 gnment. 128,140,144,146,154,156,157 The alignment consists of 66% eukaryotic ( 66% plantae) 171,172,173,175,178,181,182 sequences. (Descriptions of some sequences were not readily availa- 188,189,190,191,193,195,197 ble.) The file containing the sequence descriptions can be found in 209,212,213,214,230,231,232 the attachment, under the name 2zbjA.descr. 233

2.3 Residue ranking in 2zbjA Table 1. Clusters of top ranking residues in 2zbjA. The 2zbjA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues

2 Table 3. continued res type disruptive mutations

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

Fig. 4. Residues in 2zbjA, 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. Phosphate ion binding site. Table 2 lists the top 25% of residues Fig. 5. Residues in 2zbjA, at the interface with phosphate ion, colored by at the interface with 2zbjAPO41285 (phosphate ion). The following their relative importance. The ligand (phosphate ion) is colored green. Atoms table (Table 3) suggests possible disruptive replacements for these further than 30A˚ away from the geometric center of the ligand, as well as on residues (see Section 3.6). the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 2zbjA.) Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) Figure 5 shows residues in 2zbjA colored by their importance, at the 98 G G(85) 0.17 4/4 3.48 site interface with 2zbjAPO41285. A(3) Interface with 2zbjA3.Table 4 lists the top 25% of residues at T(2) the interface with 2zbjA3. The following table (Table 5) suggests S(3)Q possible disruptive replacements for these residues (see Section 3.6). V(1)L.K Table 4. RYE res type subst’s cvg noc/ dist (%) bb (A˚ ) Table 2. The top 25% of residues in 2zbjA at the interface with phos- 178 P P(89) 0.07 27/9 3.52 phate ion.(Field names: res: residue number in the PDB entry; type: amino R(1). 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 Q(4) the number of contacts realized through backbone atoms given in the bracket; A(2)LKE dist: distance of closest apporach to the ligand. ) 128 F F(94) 0.10 17/13 3.27 .(3)DYV MA Table 3. 175 F Y(80) 0.22 6/0 3.65 res type disruptive F(8) mutations H(9)LSW 98 G (R)(E)(K)(H) continued in next column continued in next column

3 Table 4. continued Table 6. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist antn (%) bb (A˚ ) (%) bb (A˚ ) C 10 D D(91) 0.03 5/1 2.17 site N(4) Table 4. The top 25% of residues in 2zbjA at the interface with 2zbjA3. V(1)GF (Field names: res: residue number in the PDB entry; type: amino acid type; L(2)E substs: substitutions seen in the alignment; with the percentage of each type 8 E E(86) 0.04 4/0 2.31 site in the bracket; noc/bb: number of contacts with the ligand, with the number of V(7) contacts realized through backbone atoms given in the bracket; dist: distance A(4)DFN of closest apporach to the ligand. ) Q 34 S S(82) 0.07 1/0 4.09 P(9)X Table 5. W(2) res type disruptive I(2)TDQ mutations F 178 P (Y)(T)(HR)(CG) 24 H H(83) 0.08 5/0 2.12 128 F (K)(E)(Q)(R) R(7) 175 F (K)(E)(Q)(D) .(3)LA E(1) Table 5. List of disruptive mutations for the top 25% of residues in 2zbjA, D(1) that are at the interface with 2zbjA3. Q(1)SG 19 D D(66) 0.25 5/1 2.37 site .(12) S(2) K(2) P(9)AY N(3) E(2)T

Table 6. The top 25% of residues in 2zbjA 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; dist: distance of closest apporach to the ligand. )

Table 7. res type disruptive mutations 10 D (R)(H)(FW)(Y) 8 E (H)(Y)(FW)(R) 34 S (R)(K)(H)(Q) 24 H (E)(T)(QMD)(K) 19 D (R)(H)(FW)(Y)

Fig. 6. Residues in 2zbjA, at the interface with 2zbjA3, colored by their rela- Table 7. List of disruptive mutations for the top 25% of residues in 2zbjA, tive importance. 2zbjA3 is shown in backbone representation (See Appendix that are at the interface with manganese (ii) ion. for the coloring scheme for the protein chain 2zbjA.) Figure 7 shows residues in 2zbjA colored by their importance, at the Figure 6 shows residues in 2zbjA colored by their importance, at the interface with 2zbjAMN238. interface with 2zbjA3. Calcium ion binding site. Table 8 lists the top 25% of residues Manganese (ii) ion binding site. Table 6 lists the top 25% of resi- at the interface with 2zbjACA239 (calcium ion). The following table dues at the interface with 2zbjAMN238 (manganese (ii) ion). The (Table 9) suggests possible disruptive replacements for these residues following table (Table 7) suggests possible disruptive replacements (see Section 3.6). for these residues (see Section 3.6).

4 Table 9. res type disruptive mutations 10 D (R)(H)(FW)(Y) 24 H (E)(T)(QMD)(K) 19 D (R)(H)(FW)(Y)

Table 9. List of disruptive mutations for the top 25% of residues in 2zbjA, that are at the interface with calcium ion.

Fig. 7. Residues in 2zbjA, at the interface with manganese (ii) ion, colored by their relative importance. The ligand (manganese (ii) 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 2zbjA.)

Table 8. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 10 D D(91) 0.03 4/0 2.44 site N(4) V(1)GF Fig. 8. Residues in 2zbjA, at the interface with calcium ion, colored by their L(2)E relative importance. The ligand (calcium ion) is colored green. Atoms further 24 H H(83) 0.08 2/0 4.55 than 30A˚ away from the geometric center of the ligand, as well as on the line R(7) of sight to the ligand were removed. (See Appendix for the coloring scheme .(3)LA for the protein chain 2zbjA.) E(1) D(1) Q(1)SG Figure 8 shows residues in 2zbjA colored by their importance, at the 19 D D(66) 0.25 4/0 2.32 site interface with 2zbjACA239. .(12) Interface with 2zbjA2.Table 10 lists the top 25% of residues at S(2) the interface with 2zbjA2. The following table (Table 11) suggests K(2) possible disruptive replacements for these residues (see Section 3.6). P(9)AY Table 10. N(3) res type subst’s cvg noc/ dist E(2)T (%) bb (A˚ ) 188 V V(68) 0.23 10/0 3.99 Table 8. The top 25% of residues in 2zbjA at the interface with calcium L(18) ion.(Field names: res: residue number in the PDB entry; type: amino acid N(2) type; substs: substitutions seen in the alignment; with the percentage of each M(1)I 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: T(2) distance of closest apporach to the ligand. ) E(2) A(2). continued in next column

5 Table 10. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) S(1)R

Table 10. The top 25% of residues in 2zbjA at the interface with 2zbjA2. (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. 10. A possible active surface on the chain 2zbjA. The larger cluster it belongs to is shown in blue. Table 11. res type disruptive mutations while Table 13 suggests possible disruptive replacements for these 188 V (Y)(R)(K)(E) residues (see Section 3.6).

Table 11. List of disruptive mutations for the top 25% of residues in Table 12. 2zbjA, that are at the interface with 2zbjA2. res type substitutions(%) cvg 191 F F(95)W(2)STRL 0.00 54 Y Y(89)F(9)L.S 0.02 7 V V(95).I(2)LCF 0.03 212 F F(92)L(2)A(2)WS 0.04 .V 6 A A(86)G(5).T(4)L 0.06 FHEV(1)S 190 S S(77)D(10)N(10) 0.09 G(1)TAH 52 I I(80)V(16)LM(1) 0.14 .YT 232 L L(74)I(6)V(12) 0.14 .(1)T(3)MF(1) 181 I I(50)L(32)M(3) 0.15 F(9)V(2)KAP 61 L L(83)M(2)V(1) 0.18 I(9)F(2)Q.S 214 I L(61)I(26)M(2) 0.18 F(2)V(6).A 30 K N(85)S(2)XK(4)E 0.19 D(1)G(3)H(1)RY. C 189 A A(77)S(4)T(8) 0.19 R(3)HQ(1).GL V(1)F 182 W W(66)K(3)R(15) 0.21 Fig. 9. Residues in 2zbjA, at the interface with 2zbjA2, colored by their rela- F(1)H(1)DY(3) tive importance. 2zbjA2 is shown in backbone representation (See Appendix S(1)L(1)ET(1)GQ for the coloring scheme for the protein chain 2zbjA.) VCN(1) 113 S S(80)V.(9)A(3) 0.22 L(1)T(1)M(1)CNG Figure 9 shows residues in 2zbjA colored by their importance, at the RQ interface with 2zbjA2. 85 L V(20)L(73)F(1) 0.23 2.4.3 Possible novel functional surfaces at 25% coverage. One I(1).GAMTH group of residues is conserved on the 2zbjA surface, away from (or continued in next column susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2zbj. It is shown in Fig. 10. 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 12,

6 Table 12. continued 3.2 Known substitutions res type substitutions(%) cvg One of the table columns is “substitutions” - other amino acid types 188 V V(68)L(18)N(2) 0.23 seen at the same position in the alignment. These amino acid types M(1)IT(2)E(2) may be interchangeable at that position in the protein, so if one wants A(2).S(1)R to affect the protein by a point mutation, they should be avoided. For 29 I V(67)KI(24)A 0.24 example if the substitutions are “RVK” and the original protein has L(2)XNRM(1)CDF. an R at that position, it is advisable to try anything, but RVK. Conver- 81 L L(84)V(6)QI(1) 0.25 sely, when looking for substitutions which will not affect the protein, F(1)P(1)M(2) one may try replacing, R with K, or (perhaps more surprisingly), with .(1)AS V. The percentage of times the substitution appears in the alignment 233 F F(74).(12)L(5) 0.25 is given in the immediately following bracket. No percentage is given A(1)V(3)Y(1)MD in the cases when it is smaller than 1%. This is meant to be a rough I(1)T guide - due to rounding errors these percentages often do not add up to 100%. Table 12. Residues forming surface ”patch” in 2zbjA. 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by Table 13. 2 at least 10A˚ , which is roughly the area needed for one water mole- res type disruptive cule to come in the contact with the residue. Furthermore, we require mutations that these residues form a “cluster” of residues which have neighbor 191 F (K)(E)(Q)(D) within 5A˚ from any of their heavy atoms. 54 Y (K)(Q)(MR)(E) Note, however, that, if our picture of protein evolution is correct, 7 V (R)(KE)(Y)(QD) the neighboring residues which are not surface accessible might be 212 F (K)(E)(Q)(D) equally important in maintaining the interaction specificity - they 6 A (KR)(E)(Y)(Q) should not be automatically dropped from consideration when choo- 190 S (R)(K)(QMH)(FW) sing the set for mutagenesis. (Especially if they form a cluster with 52 I (R)(Y)(H)(K) the surface residues.) 232 L (R)(Y)(H)(T) 181 I (Y)(R)(T)(H) 3.4 Number of contacts 61 L (Y)(R)(H)(T) Another column worth noting is denoted “noc/bb”; it tells the num- 214 I (YR)(TH)(K)(E) ber of contacts heavy atoms of the residue in question make across 30 K (Y)(FW)(T)(VA) the interface, as well as how many of them are realized through the 189 A (E)(K)(YR)(D) backbone atoms (if all or most contacts are through the backbone, 182 W (KE)(D)(TQ)(R) mutation presumably won’t have strong impact). Two heavy atoms 113 S (R)(K)(H)(FW) are considered to be “in contact” if their centers are closer than 5A˚ . 85 L (R)(Y)(H)(E) 188 V (Y)(R)(K)(E) 3.5 Annotation 29 I (Y)(R)(T)(H) If the residue annotation is available (either from the pdb file or 81 L (Y)(R)(H)(T) from other sources), another column, with the header “annotation” 233 F (K)(E)(R)(Q) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 13. Disruptive mutations for the surface patch in 2zbjA. bond forming residue), hb (hydrogen bond forming residue, jb (james bond forming residue), and sb (for salt bridge forming residue). 3.6 Mutation suggestions 3 NOTES ON USING TRACE RESULTS Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 3.1 Coverage they are meant to be disruptive to the interaction of the protein Trace results are commonly expressed in terms of coverage: the resi- with its ligand. The attempt is made to complement the following due is important if its “coverage” is small - that is if it belongs to properties: small [AV GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], to some nearby percentage), sorted by the strength of the presumed and NH2 group possession [NQRK]. The suggestions are listed evolutionary pressure. (I.e., the smaller the coverage, the stronger the according to how different they appear to be from the original amino pressure on the residue.) Starting from the top of that list, mutating a acid, and they are grouped in round brackets if they appear equally couple of residues should affect the protein somehow, with the exact disruptive. From left to right, each bracketed group of amino acid effects to be determined experimentally. types resembles more strongly the original (i.e. is, presumably, less

7 4.3 Credits 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number COVERAGE of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are V some percent identities. A percent pairwise alignment identity is defi- 100% 50% 30% 5% ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best V relative) for all N sequences, then finding the minimum of these N RELATIVE IMPORTANCE numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License. Fig. 11. Coloring scheme used to color residues by their relative importance. 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 4.3.3 DSSP In this work a residue is considered solvent accessi- ˚ 2 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A , which is roughly the area needed for one water molecule to come in 4.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: by [email protected] November 18,2002,

• alignment# number of the position in the alignment http://www.cmbi.kun.nl/gv/dssp/descrip.html. • residue# residue number in the PDB file • type amino acid type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- gnment as a starting point for the analysis (sequences shorter than • rank rank of the position according to older version of ET 75% of the query are taken out, however); R. Schneider, A. de • variability has two subfields: Daruvar, and C. Sander. ”The HSSP database of protein structure- 1. number of different amino acids appearing in in this column sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. of the alignment 2. their type http://swift.cmbi.kun.nl/swift/hssp/ • rho ET score - the smaller this value, the lesser variability of this position across the branches of the tree (and, presumably, 4.3.5 LaTex The text for this report was processed using LATEX; the greater the importance for the protein) Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 4.3.6 Muscle When making alignments “from scratch”, report • gaps percentage of gaps in this column maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and 4.2 Color schemes used high throughput.” Nucleic Acids Research 32(5), 1792-97. The following color scheme is used in figures with residues colored by cluster size: black is a single-residue cluster; clusters composed of http://www.drive5.com/muscle/ more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.7 Pymol The figures in this report were produced using quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, Pymol. The scripts can be found in the attachment. Pymol bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, is an open-source application copyrighted by DeLano Scien- DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tific LLC (2005). For more information about Pymol see tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. http://pymol.sourceforge.net/. (Note for Windows The colors used to distinguish the residues by the estimated users: the attached package needs to be unzipped for Pymol to read evolutionary pressure they experience can be seen in Fig. 11. the scripts and launch the viewer.)

8 4.4 Note about ET Viewer 4.7 Attachments Dan Morgan from the Lichtarge lab has developed a visualization The following files should accompany this report: tool specifically for viewing trace results. If you are interested, please • 2zbjA.complex.pdb - coordinates of 2zbjA with all of its inter- visit: acting partners http://mammoth.bcm.tmc.edu/traceview/ • 2zbjA.etvx - ET viewer input file for 2zbjA The viewer is self-unpacking and self-installing. Input files to be used • 2zbjA.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 2zbjA main report. • 2zbjA.ranks - Ranks file in sequence order for 2zbjA 4.5 Citing this work • 2zbjA.clusters - Cluster descriptions for 2zbjA The method used to rank residues and make predictions in this report • 2zbjA.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 2zbjA Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 2zbjA.descr - description of sequences used in 2zbjA msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 2zbjA.ranks sorted - full listing of residues and their ranking for of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 2zbjA tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 2zbjA.2zbjAPO41285.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 2zbjA.cbcvg - used by other 2zbjA – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 2zbjA.2zbjA3.if.pml - Pymol script for Figure 6 of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 2zbjA.2zbjAMN238.if.pml - Pymol script for Figure 7 • 2zbjA.2zbjACA239.if.pml - Pymol script for Figure 8 4.6 About report maker • 2zbjA.2zbjA2.if.pml - Pymol script for Figure 9 report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston.

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