Pages 1–9 1wmh Evolutionary trace report by report maker December 24, 2009

4 Notes on using trace results 7 4.1 Coverage 7 4.2 Known substitutions 7 4.3 Surface 7 4.4 Number of contacts 7 4.5 Annotation 8 4.6 Mutation suggestions 8

5 Appendix 8 5.1 File formats 8 5.2 Color schemes used 8 5.3 Credits 8 5.3.1 Alistat 8 5.3.2 CE 8 5.3.3 DSSP 8 5.3.4 HSSP 8 5.3.5 LaTex 9 5.3.6 Muscle 9 5.3.7 Pymol 9 5.4 Note about ET Viewer 9 5.5 Citing this work 9 5.6 About report maker 9 CONTENTS 5.7 Attachments 9

1 Introduction 1 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1wmh): 2 Chain 1wmhA 1 Title: Crystal structure of a pb1 domain complex of protein kinase c 2.1 Q5R4K9 overview 1 iota and par6 alpha 2.2 Multiple sequence alignment for 1wmhA 1 Compound: Mol id: 1; molecule: protein kinase c, iota type; chain: 2.3 Residue ranking in 1wmhA 1 a; fragment: pb1 domain; synonym: npkc-iota, atypical protein 2.4 Top ranking residues in 1wmhA and their position kinase c-lambda/iota, apkc-lambda/iota; ec: 2.7.1.37; engineered: on the structure 2 yes; mol id: 2; molecule: partitioning defective-6 homolog alpha; 2.4.1 Clustering of residues at 25% coverage. 2 chain: b; fragment: pb1 domain; synonym: par-6 alpha, par-6a, par-6, 2.4.2 Overlap with known functional surfaces at par6c, tax interaction protein 40, tip-40; engineered: yes 25% coverage. 2 Organism, scientific name: Homo Sapiens; 2.4.3 Possible novel functional surfaces at 25% 1wmh contains unique chains 1wmhA (83 residues) and 1wmhB coverage. 3 (82 residues)

3 Chain 1wmhB 4 2 CHAIN 1WMHA 3.1 Q9NPB6 overview 4 3.2 Multiple sequence alignment for 1wmhB 4 2.1 Q5R4K9 overview 3.3 Residue ranking in 1wmhB 4 From SwissProt, id Q5R4K9, 100% identical to 1wmhA: 3.4 Top ranking residues in 1wmhB and their position Description: Protein kinase C, iota type (EC 2.7.1.37) (nPKC-iota). on the structure 5 Organism, scientific name: Pongo pygmaeus (Orangutan). 3.4.1 Clustering of residues at 26% coverage. 5 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.4.2 Overlap with known functional surfaces at Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 26% coverage. 5 Catarrhini; Hominidae; Pongo. 3.4.3 Possible novel functional surfaces at 26% Function: Calcium-independent, phospholipid-dependent, serine- coverage. 6 and threonine-specific enzyme. Is not activated by phorbol esters

1 Lichtarge lab 2006 or diaglycerol. May play role in the secretory response to nutrients. Involved in cell polarization processes and the formation of epithelial tight junctions (By similarity). Catalytic activity: ATP + a protein = ADP + a phosphoprotein. Enzyme regulation: Might be a target for novel lipid activators that are elevated during nutrient-stimulated insulin secretion (By similarity). Fig. 1. Residues 16-98 in 1wmhA colored by their relative importance. (See Subunit: Interacts with PARD6A, PARD6B and PARD6G. Part Appendix, Fig.12, for the coloring scheme.) of a quaternary complex containing PARD3, some PARD6 pro- tein (PARD6A, PARD6B or PARD6G) and some GTPase protein (CDC42 or RAC1). Interacts with CENTA1 (By similarity). Pymol script for producing this figure can be found in the attachment. Similarity: Belongs to the Ser/Thr protein kinase family. PKC subfamily. Similarity: Contains 1 phorbol-ester/DAG-type zinc finger. 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 1wmhA For the chain 1wmhA, the alignment 1wmhA.msf (attached) with 31 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 1wmhA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 31 Total number of residues: 2540 Smallest: 73 Largest: 83 Average length: 81.9 Alignment length: 83 Average identity: 50% Most related pair: 99% Fig. 2. Residues in 1wmhA, colored by their relative importance. Clockwise: Most unrelated pair: 13% front, back, top and bottom views. Most distant seq: 33%

Furthermore, 3% of residues show as conserved in this alignment. 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the The alignment consists of 41% eukaryotic ( 25% vertebrata, 6% top 25% of all residues, this time colored according to clusters they arthropoda) sequences. (Descriptions of some sequences were not belong to. The clusters in Fig.3 are composed of the residues listed readily available.) The file containing the sequence descriptions can in Table 1. be found in the attachment, under the name 1wmhA.descr. Table 1. 2.3 Residue ranking in 1wmhA cluster size member color residues The 1wmhA sequence is shown in Fig. 1, with each residue colored red 19 20,29,58,60,61,63,65,67,68 according to its estimated importance. The full listing of residues in 69,73,76,77,79,80,82,83,92 1wmhA can be found in the file called 1wmhA.ranks sorted in the 95 attachment.

2.4 Top ranking residues in 1wmhA and their position Table 1. Clusters of top ranking residues in 1wmhA. on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 1wmhA colored 2.4.2 Overlap with known functional surfaces at 25% coverage. by their importance: bright red and yellow indicate more conser- The name of the ligand is composed of the source PDB identifier ved/important residues (see Appendix for the coloring scheme). A and the heteroatom name used in that file.

2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ )

Table 2. The top 25% of residues in 1wmhA at the interface with 1wmhB. (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 3. res type disruptive mutations 63 D (R)(FWH)(KYVCAG)(TQM) 67 D (R)(FWH)(KYVCAG)(TQM) 79 E (FWH)(YVCARG)(T)(SNKLPI) 61 W (K)(E)(Q)(D) 68 P (Y)(T)(HR)(SCG) 76 E (FWH)(R)(YVCAG)(T) 82 R (D)(ELPI)(FTYVMAW)(SCG) 65 E (FWH)(R)(YVCAG)(T) Fig. 3. Residues in 1wmhA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for 83 L (YR)(H)(T)(KE) the coloring scheme). Clockwise: front, back, top and bottom views. The 69 C (E)(KR)(D)(YH) corresponding Pymol script is attached. Table 3. List of disruptive mutations for the top 25% of residues in 1wmhA, that are at the interface with 1wmhB. Interface with 1wmhB.Table 2 lists the top 25% of residues at the interface with 1wmhB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 4.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 63 D D(100) 0.04 22/0 2.73 67 D D(100) 0.04 33/3 2.71 79 E E(100) 0.04 49/0 3.19 61 W W(96) 0.12 9/0 3.82 Y(3) 68 P P(96) 0.12 12/11 3.61 K(3) 76 E E(96) 0.12 38/5 2.80 D(3) 82 R R(96) 0.12 24/0 3.25 T(3) 65 E E(90) 0.18 28/1 2.62 D(9) 83 L L(83) 0.24 15/0 3.57 I(6) C(6) M(3) 69 C C(83) 0.25 29/11 3.27 I(9) Fig. 4. Residues in 1wmhA, at the interface with 1wmhB, colored by V(3) their relative importance. 1wmhB is shown in backbone representation (See R(3) Appendix for the coloring scheme for the protein chain 1wmhA.) continued in next column

Figure 4 shows residues in 1wmhA colored by their importance, at the interface with 1wmhB.

3 2.4.3 Possible novel functional surfaces at 25% coverage. One Table 4. continued group of residues is conserved on the 1wmhA surface, away from (or res type substitutions(%) cvg susbtantially larger than) other functional sites and interfaces reco- M(3) gnizable in PDB entry 1wmh. It is shown in Fig. 5. The residues 69 C C(83)I(9)V(3) 0.25 R(3)

Table 4. Residues forming surface ”patch” in 1wmhA.

Table 5. res type disruptive mutations 63 D (R)(FWH)(KYVCAG)(TQM) 67 D (R)(FWH)(KYVCAG)(TQM) 79 E (FWH)(YVCARG)(T)(SNKLPI) 58 T (R)(K)(H)(FQW) 60 K (Y)(FTW)(SVCAG)(H) 61 W (K)(E)(Q)(D) 68 P (Y)(T)(HR)(SCG) 76 E (FWH)(R)(YVCAG)(T) 77 L (YR)(TH)(SKECG)(FQWD) 82 R (D)(ELPI)(FTYVMAW)(SCG) 95 H (T)(E)(D)(CG) 80 A (KR)(YE)(QH)(D) 92 L (YR)(TH)(SKECG)(FQWD) 65 E (FWH)(R)(YVCAG)(T) 73 S (KR)(FQMWH)(NELPI)(Y) Fig. 5. A possible active surface on the chain 1wmhA. 20 K (Y)(T)(FW)(VCAG) 29 I (YR)(H)(E)(TK) 83 L (YR)(H)(T)(KE) belonging to this surface ”patch” are listed in Table 4, while Table 69 C (E)(KR)(D)(YH) 5 suggests possible disruptive replacements for these residues (see Section 4.6). Table 5. Disruptive mutations for the surface patch in 1wmhA. Table 4. res type substitutions(%) cvg 63 D D(100) 0.04 67 D D(100) 0.04 79 E E(100) 0.04 3 CHAIN 1WMHB 58 T T(96)I(3) 0.12 3.1 Q9NPB6 overview 60 K K(96)Q(3) 0.12 From SwissProt, id Q9NPB6, 100% identical to 1wmhB: 61 W W(96)Y(3) 0.12 Description: Partitioning defective-6 homolog alpha (PAR-6 alpha) 68 P P(96)K(3) 0.12 (PAR-6A) (PAR-6) (PAR6C) (Tax interaction protein 40) (TIP-40). 76 E E(96)D(3) 0.12 Organism, scientific name: Homo sapiens (Human). 77 L L(96)I(3) 0.12 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 82 R R(96)T(3) 0.12 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 95 H H(93)L(3)K(3) 0.13 Catarrhini; Hominidae; Homo. 80 A A(96)S(3) 0.14 Function: Adapter protein involved in asymmetrical cell division 92 L L(93)I(6) 0.17 and cell polarization processes. Probably involved in the formation 65 E E(90)D(9) 0.18 of epithelial tight junctions. Association with PARD3 may prevent 73 S T(19)S(80) 0.20 the interaction of PARD3 with F11R/JAM1, thereby preventing tight 20 K K(93).(3)R(3) 0.22 junction assembly. The PARD6-PARD3 complex links GTP-bound 29 I I(80)V(9)C(6) 0.23 Rho small GTPases to atypical protein kinase C proteins. N(3) Subunit: Interacts with PARD3. Interacts with GTP-bound forms of 83 L L(83)I(6)C(6) 0.24 CDC42, ARHQ/TC10 and RAC1. Interacts with the N-terminal part continued in next column of PRKCI and PRKCZ. Part of a complex with PARD3, CDC42 or RAC1 and PRKCI or PRKCZ. Interacts with human T-cell leukemia virus type I TAX protein. Interacts with PALS1. Interaction:

4 Subcellular location: Cytoplasmic; colocalizes with GTP-bound CDC42 or RAC1 at membrane ruffles and with PARD3 and PRKCI at epithelial tight junctions. Alternative products: Event=Alternative splicing; Named isoforms=2; Name=1; IsoId=Q9NPB6-1; Sequence=Displayed; Note=No experimental confirmation available; Name=2; IsoId=Q9NPB6-2; Sequence=VSP Fig. 6. Residues 14-95 in 1wmhB colored by their relative importance. (See 007459; Appendix, Fig.12, for the coloring scheme.) Tissue specificity: Expressed in pancreas, skeletal muscle, brain and heart. Weakly expressed in kidney and placenta. importance: bright red and yellow indicate more conserved/important Domain: The pseudo-CRIB domain together with the PDZ domain is residues (see Appendix for the coloring scheme). A Pymol script for required for the interaction with Rho small GTPases (By similarity). producing this figure can be found in the attachment. Similarity: Belongs to the PAR6 family. Similarity: Contains 1 PDZ (DHR) domain. Similarity: Contains 1 pseudo-CRIB domain. 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. 3.2 Multiple sequence alignment for 1wmhB For the chain 1wmhB, the alignment 1wmhB.msf (attached) with 32 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 1wmhB.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 32 Total number of residues: 2569 Smallest: 71 Largest: 82 Average length: 80.3 Alignment length: 82 Average identity: 55% Fig. 7. Residues in 1wmhB, colored by their relative importance. Clockwise: Most related pair: 99% front, back, top and bottom views. Most unrelated pair: 35% Most distant seq: 48% 3.4.1 Clustering of residues at 26% coverage. Fig. 8 shows the Furthermore, 8% of residues show as conserved in this alignment. top 26% of all residues, this time colored according to clusters they The alignment consists of 34% eukaryotic ( 21% vertebrata, 6% belong to. The clusters in Fig.8 are composed of the residues listed arthropoda) sequences. (Descriptions of some sequences were not in Table 6. readily available.) The file containing the sequence descriptions can Table 6. be found in the attachment, under the name 1wmhB.descr. cluster size member color residues 3.3 Residue ranking in 1wmhB red 12 61,63,67,68,69,70,71,72,73 The 1wmhB sequence is shown in Fig. 6, with each residue colored 76,80,88 according to its estimated importance. The full listing of residues in blue 6 24,25,26,27,28,50 1wmhB can be found in the file called 1wmhB.ranks sorted in the yellow 2 42,45 attachment. Table 6. Clusters of top ranking residues in 1wmhB. 3.4 Top ranking residues in 1wmhB and their position on the structure In the following we consider residues ranking among top 26% 3.4.2 Overlap with known functional surfaces at 26% coverage. of residues in the protein (the closest this analysis allows us to The name of the ligand is composed of the source PDB identifier get to 25%). Figure 7 shows residues in 1wmhB colored by their and the heteroatom name used in that file.

5 Table 8. continued res type disruptive mutations 45 L (TR)(YE)(K)(SCG)

Table 8. List of disruptive mutations for the top 26% of residues in 1wmhB, that are at the interface with 1wmhA.

Fig. 8. Residues in 1wmhB, 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.

Interface with 1wmhA.Table 7 lists the top 26% of residues at the interface with 1wmhA. The following table (Table 8) suggests possible disruptive replacements for these residues (see Section 4.6). Table 7. res type subst’s cvg noc/ dist Fig. 9. Residues in 1wmhB, at the interface with 1wmhA, colored by their relative importance. 1wmhA is shown in backbone representation (See (%) bb (A˚ ) Appendix for the coloring scheme for the protein chain 1wmhB.) 25 E E(100) 0.09 4/0 4.37 27 R R(100) 0.09 63/9 2.80 28 R R(100) 0.09 62/20 2.97 Figure 9 shows residues in 1wmhB colored by their importance, at 45 L L(90) 0.23 2/0 4.14 the interface with 1wmhA. F(3) Y(3) 3.4.3 Possible novel functional surfaces at 26% coverage. One R(3) group of residues is conserved on the 1wmhB surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1wmh. It is shown in Fig. 10. The residues Table 7. The top 26% of residues in 1wmhB at the interface with 1wmhA. belonging to this surface ”patch” are listed in Table 9, while Table (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 10 suggests possible disruptive replacements for these residues (see in the bracket; noc/bb: number of contacts with the ligand, with the number of Section 4.6). contacts realized through backbone atoms given in the bracket; dist: distance Table 9. of closest apporach to the ligand. ) res type substitutions(%) cvg 25 E E(100) 0.09 Table 8. 27 R R(100) 0.09 res type disruptive 28 R R(100) 0.09 mutations 50 H H(100) 0.09 25 E (FWH)(YVCARG)(T)(SNKLPI) 24 A A(87)S(9)G(3) 0.24 27 R (TD)(SYEVCLAPIG)(FMW)(N) 28 R (TD)(SYEVCLAPIG)(FMW)(N) Table 9. Residues forming surface ”patch” in 1wmhB. continued in next column

6 Fig. 10. A possible active surface on the chain 1wmhB. Fig. 11. Another possible active surface on the chain 1wmhB.

Table 10. Table 11. continued res type disruptive res type substitutions(%) cvg mutations 45 L L(90)F(3)Y(3) 0.23 25 E (FWH)(YVCARG)(T)(SNKLPI) R(3) 27 R (TD)(SYEVCLAPIG)(FMW)(N) 28 R (TD)(SYEVCLAPIG)(FMW)(N) Table 11. Residues forming surface ”patch” in 1wmhB. 50 H (E)(TQMD)(SNKVCLAPIG)(YR) 24 A (KR)(E)(Y)(QH) Table 12. Table 10. Disruptive mutations for the surface patch in 1wmhB. res type disruptive mutations Another group of surface residues is shown in Fig.11. The residues 42 F (KE)(TQD)(SNCRG)(M) belonging to this surface ”patch” are listed in Table 11, while Table 70 P (YR)(TH)(SKECG)(FQWD) 12 suggests possible disruptive replacements for these residues (see 73 N (Y)(FTWH)(SEVCARG)(MD) Section 4.6). 63 D (R)(FWH)(K)(Y) 67 D (R)(FWH)(YVCAG)(K) Table 11. 68 L (R)(TY)(KE)(SCHG) res type substitutions(%) cvg 88 L (YR)(TH)(SKECG)(FQWD) 42 F F(100) 0.09 86 P (YR)(H)(T)(KE) 70 P P(100) 0.09 71 L (YR)(TH)(SKECG)(FQWD) 73 N N(100) 0.09 76 S (R)(FKWH)(YM)(EQ) 63 D D(90)S(9) 0.11 61 Y (K)(QM)(ER)(NVLAPI) 67 D D(96)E(3) 0.12 72 T (R)(FKWH)(M)(EQ) 68 L L(96)F(3) 0.13 69 L (YR)(TH)(SKECG)(FQWD) 88 L L(96)P(3) 0.15 45 L (TR)(YE)(K)(SCG) 86 P P(93)S(3)N(3) 0.16 71 L I(90)L(9) 0.18 Table 12. Disruptive mutations for the surface patch in 1wmhB. 76 S N(90)S(9) 0.18 61 Y Y(96)X(3) 0.20 72 T N(81)T(18) 0.21 69 L L(96)P(3) 0.22 4 NOTES ON USING TRACE RESULTS continued in next column 4.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to

7 some small top percentage of residues [100% is all of the residues in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the COVERAGE pressure on the residue.) Starting from the top of that list, mutating a

couple of residues should affect the protein somehow, with the exact V effects to be determined experimentally. 100% 50% 30% 5% 4.2 Known substitutions One of the table columns is “substitutions” - other amino acid types 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 to affect the protein by a point mutation, they should be avoided. For V example if the substitutions are “RVK” and the original protein has RELATIVE IMPORTANCE 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 Fig. 12. Coloring scheme used to color residues by their relative importance. 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 guide - due to rounding errors these percentages often do not add up properties: small [AV GSTC], medium [LPNQDEMIK], large to 100%. [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- tively [KHR], or negatively [DE] charged, aromatic [WFYH], 4.3 Surface long aliphatic chain [EKRQM], OH-group possession [SDETY ], To detect candidates for novel functional interfaces, first we look for and NH2 group possession [NQRK]. The suggestions are listed residues that are solvent accessible (according to DSSP program) by according to how different they appear to be from the original amino 2 at least 10A˚ , which is roughly the area needed for one water mole- acid, and they are grouped in round brackets if they appear equally cule to come in the contact with the residue. Furthermore, we require disruptive. From left to right, each bracketed group of amino acid that these residues form a “cluster” of residues which have neighbor types resembles more strongly the original (i.e. is, presumably, less within 5A˚ from any of their heavy atoms. disruptive) These suggestions are tentative - they might prove disrup- Note, however, that, if our picture of protein evolution is correct, tive to the fold rather than to the interaction. Many researcher will the neighboring residues which are not surface accessible might be choose, however, the straightforward alanine mutations, especially in equally important in maintaining the interaction specificity - they the beginning stages of their investigation. should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 5 APPENDIX 4.4 Number of contacts 5.1 File formats Another column worth noting is denoted “noc/bb”; it tells the num- Files with extension “ranks sorted” are the actual trace results. The ber of contacts heavy atoms of the residue in question make across fields in the table in this file: the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, • mutation presumably won’t have strong impact). Two heavy atoms alignment# number of the position in the alignment are considered to be “in contact” if their centers are closer than 5A˚ . • residue# residue number in the PDB file • type amino acid type 4.5 Annotation • rank rank of the position according to older version of ET If the residue annotation is available (either from the pdb file or • from other sources), another column, with the header “annotation” variability has two subfields: appears. Annotations carried over from PDB are the following: site 1. number of different amino acids appearing in in this column (indicating existence of related site record in PDB ), S-S (disulfide of the alignment bond forming residue), hb (hydrogen bond forming residue, jb (james 2. their type bond forming residue), and sb (for salt bridge forming residue). • rho ET score - the smaller this value, the lesser variability of 4.6 Mutation suggestions this position across the branches of the tree (and, presumably, the greater the importance for the protein) Mutation suggestions are completely heuristic and based on comple- • cvg coverage - percentage of the residues on the structure which mentarity with the substitutions found in the alignment. Note that have this rho or smaller they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following • gaps percentage of gaps in this column

8 5.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 5.3.7 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 5.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 12. Dan Morgan from the Lichtarge lab has developed a visualization 5.3 Credits tool specifically for viewing trace results. If you are interested, please 5.3.1 Alistat alistat reads a multiple sequence alignment from the visit: 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 of residues, the average and range of the sequence lengths, and the The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two 5.5 Citing this work sequences. The ”average percent identity”, ”most related pair”, and The method used to rank residues and make predictions in this report ”most unrelated pair” of the alignment are the average, maximum, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant Evolution-Entropy Hybrid Methods for Ranking of Protein Residues seq” is calculated by finding the maximum pairwise identity (best by Importance” J. Mol. Bio. 336: 1265-82. For the original version relative) for all N sequences, then finding the minimum of these N of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- numbers (hence, the most outlying sequence). alistat is copyrighted tionary Trace Method Defines Binding Surfaces Common to Protein by HHMI/Washington University School of Medicine, 1992-2001, Families” J. Mol. Bio. 257: 342-358. and freely distributed under the GNU General Public License. report maker itself is described in Mihalek I., I. Res and O. 5.3.2 CE To map ligand binding sites from different Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type source structures, report maker uses the CE program: of service for comparative analysis of proteins.” Bioinformatics http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 22:1656-7. ”Protein structure alignment by incremental combinatorial extension 5.6 About report maker (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- 5.3.3 DSSP In this work a residue is considered solvent accessi- king visualization program was written by Ivica Res.ˇ report maker 2 ble if the DSSP program finds it exposed to water by at least 10A˚ , is copyrighted by Lichtarge Lab, Baylor College of Medicine, which is roughly the area needed for one water molecule to come in Houston. the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 5.7 Attachments by [email protected] November 18,2002, The following files should accompany this report: http://www.cmbi.kun.nl/gv/dssp/descrip.html. • 1wmhA.complex.pdb - coordinates of 1wmhA with all of its 5.3.4 HSSP Whenever available, report maker uses HSSP ali- interacting partners gnment as a starting point for the analysis (sequences shorter than • 1wmhA.etvx - ET viewer input file for 1wmhA 75% of the query are taken out, however); R. Schneider, A. de • 1wmhA.cluster report.summary - Cluster report summary for Daruvar, and C. Sander. ”The HSSP database of protein structure- 1wmhA sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 1wmhA.ranks - Ranks file in sequence order for 1wmhA http://swift.cmbi.kun.nl/swift/hssp/ • 1wmhA.clusters - Cluster descriptions for 1wmhA • 1wmhA.msf - the multiple sequence alignment used for the 5.3.5 LaTex The text for this report was processed using LAT X; E chain 1wmhA Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • 1wmhA.descr - description of sequences used in 1wmhA msf • 1wmhA.ranks sorted - full listing of residues and their ranking 5.3.6 Muscle When making alignments “from scratch”, report for 1wmhA maker uses Muscle alignment program: Edgar, Robert C. (2004), • ”MUSCLE: multiple sequence alignment with high accuracy and 1wmhA.1wmhB.if.pml - Pymol script for Figure 4 high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1wmhA.cbcvg - used by other 1wmhA – related pymol scripts

9 • 1wmhB.complex.pdb - coordinates of 1wmhB with all of its • 1wmhB.msf - the multiple sequence alignment used for the interacting partners chain 1wmhB • 1wmhB.etvx - ET viewer input file for 1wmhB • 1wmhB.descr - description of sequences used in 1wmhB msf • 1wmhB.cluster report.summary - Cluster report summary for • 1wmhB.ranks sorted - full listing of residues and their ranking 1wmhB for 1wmhB • 1wmhB.ranks - Ranks file in sequence order for 1wmhB • 1wmhB.1wmhA.if.pml - Pymol script for Figure 9 • 1wmhB.clusters - Cluster descriptions for 1wmhB • 1wmhB.cbcvg - used by other 1wmhB – related pymol scripts

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