Pages 1–9 1mvq Evolutionary trace report by report maker May 9, 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 9 4.4 Note about ET Viewer 9 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 1mvq): Title: Cratylia mollis lectin (isoform 1) in complex with methyl- alpha-d-mannose Compound: Mol id: 1; molecule: lectin, isoform 1; chain: a Organism, scientific name: Cratylia Mollis; 1mvq contains a single unique chain 1mvqA (236 residues long).

2 CHAIN 1MVQA CONTENTS 2.1 P83721 overview 1 Introduction 1 From SwissProt, id P83721, 99% identical to 1mvqA: Description: Mannose/glucose-specific lectin Cramoll (Iso1) [Con- 2 Chain 1mvqA 1 tains: Cramoll alpha chain; Cramoll beta chain] (Fragments). 2.1 P83721 overview 1 Organism, scientific name: Cratylia mollis (Camaratu bean). 2.2 Multiple sequence alignment for 1mvqA 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.3 Residue ranking in 1mvqA 1 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.4 Top ranking residues in 1mvqA and their position on eudicotyledons; rosids; eurosids I; Fabales; Fabaceae; Papilionoi- the structure 2 deae; Phaseoleae; Cratylia. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Glucose/D-mannose specific lectin. 2.4.2 Overlap with known functional surfaces at Subunit: Homotetramer. 25% coverage. 2 Ptm: The alpha and beta chains are produced by partial proteolytic 2.4.3 Possible novel functional surfaces at 25% processing of the lectin precursor by an asparaginyl endopeptidase. coverage. 5 Miscellaneous: Binds one manganese (or other transition metal) ion and one calcium ion. The metal ions are essential for the saccharide- 3 Notes on using trace results 7 binding and cell-agglutinating activities. 3.1 Coverage 7 Similarity: Belongs to the leguminous lectin family. 3.2 Known substitutions 7 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 7 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 7 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 8 and this statement is not removed.

4 Appendix 8 2.2 Multiple sequence alignment for 1mvqA 4.1 File formats 8 For the chain 1mvqA, the alignment 1mvqA.msf (attached) with 23 4.2 Color schemes used 8 sequences was used. The alignment was downloaded from the HSSP 4.3 Credits 8 database, and fragments shorter than 75% of the query as well as

1 Lichtarge lab 2006 Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 1-118 in 1mvqA colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.)

Fig. 2. Residues 119-236 in 1mvqA colored by their relative importance. (See Appendix, Fig.12, for the coloring scheme.) duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1mvqA.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1mvqA, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 23 Total number of residues: 5108 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 194 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: 222.1 in Table 1. Alignment length: 236 Table 1. Average identity: 37% cluster size member Most related pair: 99% color residues Most unrelated pair: 18% red 49 6,7,8,9,10,11,14,19,20,24,26 Most distant seq: 31% 27,34,40,52,54,56,60,61,62 63,75,76,77,79,80,81,85,86 87,89,92,93,94,95,97,98,102 Furthermore, 2% of residues show as conserved in this alignment. 104,108,109,111,113,165,171 The alignment consists of 39% eukaryotic ( 39% plantae) 172,178,190,229 sequences. (Descriptions of some sequences were not readily availa- blue 3 29,30,31 ble.) The file containing the sequence descriptions can be found in yellow 2 159,162 the attachment, under the name 1mvqA.descr. green 2 49,50 2.3 Residue ranking in 1mvqA Table 1. The 1mvqA sequence is shown in Figs. 1–2, with each residue colo- Clusters of top ranking residues in 1mvqA. red according to its estimated importance. The full listing of residues in 1mvqA can be found in the file called 1mvqA.ranks sorted in the 2.4.2 Overlap with known functional surfaces at 25% coverage. attachment. The name of the ligand is composed of the source PDB identifier 2.4 Top ranking residues in 1mvqA and their position and the heteroatom name used in that file. Calcium ion binding site. Table 2 lists the top 25% of residues at on the structure the interface with 1mvqACA237 (calcium ion). The following table In the following we consider residues ranking among top 25% of (Table 3) suggests possible disruptive replacements for these residues residues in the protein . Figure 3 shows residues in 1mvqA colored (see Section 3.6). by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A

2 Fig. 4. Residues in 1mvqA, colored according to the cluster they belong to: Fig. 5. Residues in 1mvqA, at the interface with calcium ion, colored by their red, followed by blue and yellow are the largest clusters (see Appendix for relative importance. The ligand (calcium ion) is colored green. Atoms further the coloring scheme). Clockwise: front, back, top and bottom views. The than 30A˚ away from the geometric center of the ligand, as well as on the line corresponding Pymol script is attached. of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1mvqA.)

Table 2. res type subst’s cvg noc/ dist Figure 5 shows residues in 1mvqA colored by their importance, at (%) bb (A˚ ) the interface with 1mvqACA237. 10 D D(100) 0.03 4/0 2.42 Interface with 1mvqA2.Table 4 lists the top 25% of residues at 24 H H(100) 0.03 2/0 4.64 the interface with 1mvqA2. The following table (Table 5) suggests 14 N N(95) 0.05 6/2 2.39 possible disruptive replacements for these residues (see Section 3.6). D(4) 19 D D(86) 0.14 4/0 2.40 Table 4. N(4) res type subst’s cvg noc/ dist Y(4) (%) bb (A˚ ) K(4) 87 E E(82) 0.11 1/0 4.69 A(4) Table 2. The top 25% of residues in 1mvqA at the interface with calcium D(13) 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 Table 4. The top 25% of residues in 1mvqA at the interface with type in the bracket; noc/bb: number of contacts with the ligand, with the num- 1mvqA2. (Field names: res: residue number in the PDB entry; type: amino ber of contacts realized through backbone atoms given in the bracket; dist: acid type; substs: substitutions seen in the alignment; with the percentage of distance of closest apporach to the ligand. ) 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 3. res type disruptive mutations 10 D (R)(FWH)(KYVCAG)(TQM) Table 5. 24 H (E)(TQMD)(SNKVCLAPIG)(YR) res type disruptive 14 N (Y)(FWH)(TR)(VCAG) mutations 19 D (R)(FW)(H)(VA) 87 E (H)(FW)(R)(Y)

Table 3. List of disruptive mutations for the top 25% of residues in Table 5. List of disruptive mutations for the top 25% of residues in 1mvqA, that are at the interface with calcium ion. 1mvqA, that are at the interface with 1mvqA2.

3 Table 7. res type disruptive mutations 14 N (Y)(FWH)(TR)(VCAG) 97 T (K)(FW)(MR)(H) 98 G (FWR)(H)(K)(E)

Table 7. List of disruptive mutations for the top 25% of residues in 1mvqA, that are at the interface with o1-methyl-mannose.

Fig. 6. Residues in 1mvqA, at the interface with 1mvqA2, colored by their relative importance. 1mvqA2 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1mvqA.)

Figure 6 shows residues in 1mvqA colored by their importance, at the interface with 1mvqA2. O1-methyl-mannose binding site. Table 6 lists the top 25% of residues at the interface with 1mvqMMA1 (o1-methyl-mannose). The following table (Table 7) suggests possible disruptive replace- ments for these residues (see Section 3.6). Table 6. res type subst’s cvg noc/ dist Fig. 7. Residues in 1mvqA, at the interface with o1-methyl-mannose, colo- (%) bb (A˚ ) red by their relative importance. The ligand (o1-methyl-mannose) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, 14 N N(95) 0.05 7/0 3.00 as well as on the line of sight to the ligand were removed. (See Appendix for D(4) the coloring scheme for the protein chain 1mvqA.) 97 T T(86) 0.10 1/1 4.52 S(4) R(8) Figure 7 shows residues in 1mvqA colored by their importance, at 98 G G(82) 0.16 17/17 3.27 the interface with 1mvqMMA1. K(8) Manganese (ii) ion binding site. Table 8 lists the top 25% of resi- T(4) dues at the interface with 1mvqAMN238 (manganese (ii) ion). The E(4) following table (Table 9) suggests possible disruptive replacements for these residues (see Section 3.6). Table 6. The top 25% of residues in 1mvqA at the interface with o1- Table 8. methyl-mannose.(Field names: res: residue number in the PDB entry; type: res type subst’s cvg noc/ dist amino acid type; substs: substitutions seen in the alignment; with the percen- ˚ tage of each type in the bracket; noc/bb: number of contacts with the ligand, (%) bb (A) with the number of contacts realized through backbone atoms given in the 8 E E(100) 0.03 4/0 2.08 bracket; dist: distance of closest apporach to the ligand. ) 10 D D(100) 0.03 4/0 2.08 24 H H(100) 0.03 5/0 2.10 34 S S(95) 0.06 1/0 4.26 X(4) 19 D D(86) 0.14 5/1 2.09 continued in next column

4 Table 8. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) N(4) Y(4) K(4) 32 I V(21) 0.25 1/1 4.87 I(56) M(21)

Table 8. The top 25% of residues in 1mvqA 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 9. res type disruptive mutations 8 E (FWH)(YVCARG)(T)(SNKLPI) 10 D (R)(FWH)(KYVCAG)(TQM) Fig. 8. Residues in 1mvqA, at the interface with manganese (ii) ion, colored 24 H (E)(TQMD)(SNKVCLAPIG)(YR) by their relative importance. The ligand (manganese (ii) ion) is colored green. 34 S (KR)(FQMWH)(YE)(NLPI) Atoms further than 30A˚ away from the geometric center of the ligand, as well 19 D (R)(FW)(H)(VA) as on the line of sight to the ligand were removed. (See Appendix for the 32 I (Y)(R)(H)(T) coloring scheme for the protein chain 1mvqA.)

Table 9. List of disruptive mutations for the top 25% of residues in 1mvqA, that are at the interface with manganese (ii) ion. Table 10. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) Figure 8 shows residues in 1mvqA colored by their importance, at 62 S S(78) 0.18 34/2 3.34 the interface with 1mvqAMN238. .(8) Interface with 1mvqA1.Table 10 lists the top 25% of residues at A(4) the interface with 1mvqA1. The following table (Table 11) suggests T(8) possible disruptive replacements for these residues (see Section 3.6). 76 S Y(21) 0.23 5/0 4.10 Table 10. N(4) res type subst’s cvg noc/ dist S(69) (%) bb (A˚ ) W(4) 109 W W(95) 0.06 4/4 4.00 49 T D(8) 0.24 33/2 3.05 A(4) .(4) 60 R N(47) 0.12 63/2 2.15 T(47) .(4) H(26) R(34) N(13) I(13) 75 V L(60) 0.14 2/2 4.73 Table 10. The top 25% of residues in 1mvqA at the interface with V(39) 1mvqA1. (Field names: res: residue number in the PDB entry; type: amino 108 S S(82) 0.15 11/3 3.22 acid type; substs: substitutions seen in the alignment; with the percentage of T(4) 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; Y(8) dist: distance of closest apporach to the ligand. ) G(4) 63 A V(47) 0.16 4/4 3.89 .(4) Table 11. A(43) res type disruptive M(4) mutations continued in next column continued in next column

5 Table 11. continued res type disruptive mutations 109 W (KE)(QD)(TR)(N) 60 R (T)(YD)(SCG)(EVA) 75 V (YR)(KE)(H)(QD) 108 S (K)(R)(QM)(FWH) 63 A (Y)(R)(KE)(H) 62 S (KR)(QH)(FMW)(NE) 76 S (K)(R)(QM)(E) 49 T (R)(K)(M)(FW)

Fig. 10. A possible active surface on the chain 1mvqA. The larger cluster it Table 11. List of disruptive mutations for the top 25% of residues in belongs to is shown in blue. 1mvqA, that are at the interface with 1mvqA1.

Table 12. res type substitutions(%) cvg 10 D D(100) 0.03 14 N N(95)D(4) 0.05 20 P P(86)Q(4)A(4) 0.13 D(4) 19 D D(86)N(4)Y(4) 0.14 K(4) 11 T I(4)T(65)S(30) 0.19 40 W W(73)Q(4)L(8) 0.20 P(4)S(8)

Table 12. Residues forming surface ”patch” in 1mvqA.

Table 13. res type disruptive mutations 10 D (R)(FWH)(KYVCAG)(TQM) 14 N (Y)(FWH)(TR)(VCAG) 20 P (Y)(R)(H)(T) 19 D (R)(FW)(H)(VA) 11 T (R)(K)(H)(FQW) 40 W (KE)(T)(R)(D) Fig. 9. Residues in 1mvqA, at the interface with 1mvqA1, colored by their relative importance. 1mvqA1 is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1mvqA.) Table 13. Disruptive mutations for the surface patch in 1mvqA.

Another group of surface residues is shown in Fig.11. The right panel Figure 9 shows residues in 1mvqA colored by their importance, at shows (in blue) the rest of the larger cluster this surface belongs to. the interface with 1mvqA1. The residues belonging to this surface ”patch” are listed in Table 14, 2.4.3 Possible novel functional surfaces at 25% coverage. One while Table 15 suggests possible disruptive replacements for these group of residues is conserved on the 1mvqA surface, away from (or residues (see Section 3.6). susbtantially larger than) other functional sites and interfaces reco- Table 14. gnizable in PDB entry 1mvq. It is shown in Fig. 10. The right panel res type substitutions(%) cvg shows (in blue) the rest of the larger cluster this surface belongs to. 7 V V(100) 0.03 The residues belonging to this surface ”patch” are listed in Table 12, 79 V V(95)I(4) 0.03 while Table 13 suggests possible disruptive replacements for these 85 L L(100) 0.03 residues (see Section 3.6). 86 P P(100) 0.03 31 S S(95)G(4) 0.04 54 Y Y(95).(4) 0.05 continued in next column

6 Table 15. res type disruptive mutations 7 V (KYER)(QHD)(N)(FTMW) 79 V (YR)(KE)(H)(QD) 85 L (YR)(TH)(SKECG)(FQWD) 86 P (YR)(TH)(SKECG)(FQWD) 31 S (KR)(FQMWH)(E)(NYLPI) 54 Y (K)(QM)(NVLAPI)(ER) 61 L (YR)(TH)(SCG)(KE) 34 S (KR)(FQMWH)(YE)(NLPI) 80 D (R)(FWH)(Y)(VCAG) Fig. 11. Another possible active surface on the chain 1mvqA. The larger 81 L (Y)(R)(TH)(SCG) cluster it belongs to is shown in blue. 87 E (H)(FW)(R)(Y) 113 S (R)(K)(H)(FW) Table 14. continued 60 R (T)(YD)(SCG)(EVA) res type substitutions(%) cvg 77 Y (K)(Q)(MR)(E) 61 L L(95).(4) 0.05 75 V (YR)(KE)(H)(QD) 34 S S(95)X(4) 0.06 29 I (Y)(R)(H)(T) 80 D D(91)N(8) 0.07 63 A (Y)(R)(KE)(H) 81 L L(95)M(4) 0.08 178 V (YR)(KE)(H)(QD) 87 E E(82)A(4)D(13) 0.11 62 S (KR)(QH)(FMW)(NE) 113 S S(91)P(4)G(4) 0.11 56 S (R)(K)(H)(FW) 60 R N(47).(4)R(34) 0.12 52 I (Y)(R)(H)(T) I(13) 190 F (KE)(TQD)(R)(SNCG) 77 Y Y(86)S(4)T(4) 0.13 76 S (K)(R)(QM)(E) A(4) 30 K (Y)(FTW)(SVA)(CDG) 75 V L(60)V(39) 0.14 49 T (R)(K)(M)(FW) 29 I I(69)V(17)X(4) 0.15 50 A (KR)(E)(Y)(QH) L(4)N(4) 63 A V(47).(4)A(43) 0.16 Table 15. Disruptive mutations for the surface patch in 1mvqA. M(4) 178 V V(78)L(13).(4) 0.17 I(4) 3 NOTES ON USING TRACE RESULTS 62 S S(78).(8)A(4) 0.18 T(8) 3.1 Coverage 56 S S(73).(4)N(4) 0.19 Trace results are commonly expressed in terms of coverage: the resi- G(4)A(13) due is important if its “coverage” is small - that is if it belongs to 52 I I(69).(4)V(26) 0.20 some small top percentage of residues [100% is all of the residues 190 F L(39)F(47).(4) 0.21 in a chain], according to trace. The ET results are presented in the V(8) form of a table, usually limited to top 25% percent of residues (or 76 S Y(21)N(4)S(69) 0.23 to some nearby percentage), sorted by the strength of the presumed W(4) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 30 K N(52)K(30)X(4) 0.24 pressure on the residue.) Starting from the top of that list, mutating a G(4)H(8) couple of residues should affect the protein somehow, with the exact 49 T D(8).(4)T(47) 0.24 effects to be determined experimentally. H(26)N(13) 3.2 Known substitutions 50 A A(65).(4)V(26) 0.25 T(4) One of the table columns is “substitutions” - other amino acid types seen at the same position in the alignment. These amino acid types Table 14. Residues forming surface ”patch” in 1mvqA. may be interchangeable at that position in the protein, so if one wants to affect the protein by a point mutation, they should be avoided. For example if the substitutions are “RVK” and the original protein has 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 in the cases when it is smaller than 1%. This is meant to be a rough

7 guide - due to rounding errors these percentages often do not add up to 100%. 3.3 Surface To detect candidates for novel functional interfaces, first we look for COVERAGE 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 100% 50% 30% 5% that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, 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- V sing the set for mutagenesis. (Especially if they form a cluster with RELATIVE IMPORTANCE the surface residues.) 3.4 Number of contacts Fig. 12. Coloring scheme used to color residues by their relative importance. Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across 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 appears. Annotations carried over from PDB are the following: site ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 3.6 Mutation suggestions • gaps percentage of gaps in this column Mutation suggestions are completely heuristic and based on comple- mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein The following color scheme is used in figures with residues colored with its ligand. The attempt is made to complement the following by cluster size: black is a single-residue cluster; clusters composed of properties: small [AV GSTC], medium [LPNQDEMIK], large more than one residue colored according to this hierarchy (ordered [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- by descending size): red, blue, yellow, green, purple, azure, tur- tively [KHR], or negatively [DE] charged, aromatic [WFYH], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, long aliphatic chain [EKRQM], OH-group possession [SDETY ], bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, and NH2 group possession [NQRK]. The suggestions are listed DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, according to how different they appear to be from the original amino tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. acid, and they are grouped in round brackets if they appear equally The colors used to distinguish the residues by the estimated disruptive. From left to right, each bracketed group of amino acid evolutionary pressure they experience can be seen in Fig. 12. types resembles more strongly the original (i.e. is, presumably, less disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 4 APPENDIX alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant

8 seq” is calculated by finding the maximum pairwise identity (best http://mammoth.bcm.tmc.edu/traceview/ relative) for all N sequences, then finding the minimum of these N The viewer is self-unpacking and self-installing. Input files to be used numbers (hence, the most outlying sequence). alistat is copyrighted with ETV (extension .etvx) can be found in the attachment to the by HHMI/Washington University School of Medicine, 1992-2001, main report. and freely distributed under the GNU General Public License. 4.5 Citing this work 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: The method used to rank residues and make predictions in this report http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”Protein structure alignment by incremental combinatorial extension Evolution-Entropy Hybrid Methods for Ranking of Protein Residues (CE) of the optimal path . Protein Engineering 11(9) 739-747. by Importance” J. Mol. Bio. 336: 1265-82. For the original version of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 tionary Trace Method Defines Binding Surfaces Common to Protein ble if the DSSP program finds it exposed to water by at least 10A˚ , Families” J. Mol. Bio. 257: 342-358. which is roughly the area needed for one water molecule to come in report maker itself is described in Mihalek I., I. Res and O. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version of service for comparative analysis of proteins.” Bioinformatics by [email protected] November 18,2002, 22:1656-7. http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.6 About report maker 4.3.4 HSSP Whenever available, report maker uses HSSP ali- report maker was written in 2006 by Ivana Mihalek. The 1D ran- gnment as a starting point for the analysis (sequences shorter than king visualization program was written by Ivica Res.ˇ report maker 75% of the query are taken out, however); R. Schneider, A. de is copyrighted by Lichtarge Lab, Baylor College of Medicine, Daruvar, and C. Sander. ”The HSSP database of protein structure- Houston. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 4.7 Attachments http://swift.cmbi.kun.nl/swift/hssp/ The following files should accompany this report: • 1mvqA.complex.pdb - coordinates of 1mvqA with all of its 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- interacting partners Wesley,” Reading, Mass. (1986). • 1mvqA.etvx - ET viewer input file for 1mvqA • 4.3.6 Muscle When making alignments “from scratch”, report 1mvqA.cluster report.summary - Cluster report summary for maker uses Muscle alignment program: Edgar, Robert C. (2004), 1mvqA ”MUSCLE: multiple sequence alignment with high accuracy and • 1mvqA.ranks - Ranks file in sequence order for 1mvqA high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1mvqA.clusters - Cluster descriptions for 1mvqA http://www.drive5.com/muscle/ • 1mvqA.msf - the multiple sequence alignment used for the chain 1mvqA 4.3.7 Pymol The figures in this report were produced using • 1mvqA.descr - description of sequences used in 1mvqA msf Pymol. The scripts can be found in the attachment. Pymol • is an open-source application copyrighted by DeLano Scien- 1mvqA.ranks sorted - full listing of residues and their ranking tific LLC (2005). For more information about Pymol see for 1mvqA http://pymol.sourceforge.net/. (Note for Windows • 1mvqA.1mvqACA237.if.pml - Pymol script for Figure 5 users: the attached package needs to be unzipped for Pymol to read • 1mvqA.cbcvg - used by other 1mvqA – related pymol scripts the scripts and launch the viewer.) • 1mvqA.1mvqA2.if.pml - Pymol script for Figure 6 4.4 Note about ET Viewer • 1mvqA.1mvqMMA1.if.pml - Pymol script for Figure 7 Dan Morgan from the Lichtarge lab has developed a visualization • 1mvqA.1mvqAMN238.if.pml - Pymol script for Figure 8 tool specifically for viewing trace results. If you are interested, please • 1mvqA.1mvqA1.if.pml - Pymol script for Figure 9 visit:

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