Pages 1–8 1sl8 Evolutionary trace report by report maker June 25, 2009

4.3.1 Alistat 6 4.3.2 CE 7 4.3.3 DSSP 7 4.3.4 HSSP 7 4.3.5 LaTex 7 4.3.6 Muscle 7 4.3.7 Pymol 7 4.4 Note about ET Viewer 7 4.5 Citing this work 7 4.6 About report maker 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1sl8): Title: Calcium-loaded apo-aequorin from victoria Compound: Mol id: 1; molecule: aequorin 1; chain: a; engineered: yes Organism, scientific name: ; 1sl8 contains a single unique chain 1sl8A (181 residues long).

2 CHAIN 1SL8A CONTENTS 2.1 P07164 overview

1 Introduction 1 From SwissProt, id P07164, 99% identical to 1sl8A: Description: Aequorin-1 precursor. 2 Chain 1sl8A 1 Organism, scientific name: Aequorea victoria (Jellyfish). 2.1 P07164 overview 1 : Eukaryota; Metazoa; ; ; Hydroida; 2.2 Multiple sequence alignment for 1sl8A 1 Leptomedusae; Aequoreidae; Aequorea. 2.3 Residue ranking in 1sl8A 1 Function: Ca(2+)-dependent bioluminescence photoprotein. Dis- 2.4 Top ranking residues in 1sl8A and their position on plays an emission peak at 470 nm (blue light). Trace amounts of the structure 1 calcium ion trigger the intramolecular oxidation of the chromophore, 2.4.1 Clustering of residues at 25% coverage. 2 coelenterazine into coelenteramide and CO(2) with the concomitant 2.4.2 Overlap with known functional surfaces at emission of light. 25% coverage. 2 Ptm: The reduction of the disulfide bond is necessary to regenerate 2.4.3 Possible novel functional surfaces at 25% aequorin from apoaequorin. coverage. 4 Biotechnology: Aequorin is used as an intracellular Ca(2+) indicator. Aequorin has a number of advantages over other Ca(2+) indica- 3 Notes on using trace results 6 tors, for example, low leakage rate from cells, lack of intracellular 3.1 Coverage 6 compartmentalization or sequestration and it does not disrupt cell 3.2 Known substitutions 6 functions or embryo development. 3.3 Surface 6 Similarity: Belongs to the aequorin family. 3.4 Number of contacts 6 Similarity: Contains 4 EF-hand domains. 3.5 Annotation 6 Caution: Was originally (Ref.4) thought to have a internal disulfide 3.6 Mutation suggestions 6 bond. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 6 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 6 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 6 no restrictions on its use as long as its content is in no way modified 4.3 Credits 6 and this statement is not removed.

1 Lichtarge lab 2006 Fig. 1. Residues 11-191 in 1sl8A colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

2.2 Multiple sequence alignment for 1sl8A For the chain 1sl8A, the alignment 1sl8A.msf (attached) with 34 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 1sl8A.msf. Its statistics, from the alistat program are the following: Fig. 2. Residues in 1sl8A, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 34 Total number of residues: 5224 Smallest: 136 Largest: 181 Average length: 153.6 Alignment length: 181 Average identity: 54% Most related pair: 99% Most unrelated pair: 23% Most distant seq: 48%

Furthermore, 6% of residues show as conserved in this alignment. The alignment consists of 44% eukaryotic ( 2% vertebrata, 5% fungi, 8% plantae) sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1sl8A.descr. 2.3 Residue ranking in 1sl8A The 1sl8A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1sl8A can be found in the file called 1sl8A.ranks sorted in the attachment. 2.4 Top ranking residues in 1sl8A and their position on Fig. 3. Residues in 1sl8A, colored according to the cluster they belong to: the structure red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The In the following we consider residues ranking among top 25% of resi- corresponding Pymol script is attached. dues in the protein . Figure 2 shows residues in 1sl8A colored by their importance: bright red and yellow indicate more conserved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed in Table 1.

2 Table 1. cluster size member color residues red 24 46,50,59,60,115,117,119,120 121,124,126,128,132,134,146 150,151,154,155,157,159,160 166,167 blue 18 14,17,18,19,21,22,25,26,28 31,33,37,65,66,67,81,84,88 yellow 2 139,140

Table 1. Clusters of top ranking residues in 1sl8A.

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 1sl8CA670 (calcium ion). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. Fig. 4. Residues in 1sl8A, at the interface with calcium ion, colored by their res type subst’s cvg noc/ dist relative importance. The ligand (calcium ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line (%) bb (A˚ ) of sight to the ligand were removed. (See Appendix for the coloring scheme 31 G G(100) 0.07 1/1 4.77 for the protein chain 1sl8A.) 33 I I(100) 0.07 2/2 4.25 37 E E(100) 0.07 4/0 2.43 26 D D(97) 0.08 7/3 2.16 (Table 5) suggests possible disruptive replacements for these residues Q(2) (see Section 3.6). 28 N D(55) 0.21 7/3 2.36 N(41) Table 4. A(2) res type subst’s cvg noc/ dist (%) bb (A˚ ) Table 2. The top 25% of residues in 1sl8A at the interface with calcium 120 K K(100) 0.07 1/1 4.72 ion.(Field names: res: residue number in the PDB entry; type: amino acid 124 G G(100) 0.07 1/1 4.86 type; substs: substitutions seen in the alignment; with the percentage of each 121 D D(97) 0.08 7/3 2.27 type in the bracket; noc/bb: number of contacts with the ligand, with the num- N(2) ber of contacts realized through backbone atoms given in the bracket; dist: 126 I I(97) 0.09 3/3 4.36 distance of closest apporach to the ligand. ) V(2) 119 D D(97) 0.13 7/3 2.24 G(2) Table 3. res type disruptive Table 4. The top 25% of residues in 1sl8A at the interface with calcium mutations ion.(Field names: res: residue number in the PDB entry; type: amino acid 31 G (KER)(FQMWHD)(NYLPI)(SVA) 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- 33 I (YR)(TH)(SKECG)(FQWD) ber of contacts realized through backbone atoms given in the bracket; dist: 37 E (FWH)(YVCARG)(T)(SNKLPI) distance of closest apporach to the ligand. ) 26 D (FWHR)(Y)(VCAG)(T) 28 N (Y)(H)(FW)(R) Table 5. Table 3. List of disruptive mutations for the top 25% of residues in 1sl8A, res type disruptive that are at the interface with calcium ion. mutations 120 K (Y)(FTW)(SVCAG)(HD) Figure 4 shows residues in 1sl8A colored by their importance, at the 124 G (KER)(FQMWHD)(NYLPI)(SVA) interface with 1sl8CA670. 121 D (R)(FWH)(Y)(VCAG) Calcium ion binding site. Table 4 lists the top 25% of residues continued in next column at the interface with 1sl8CA671 (calcium ion). The following table

3 Table 5. continued Table 6. continued res type disruptive res type subst’s cvg noc/ dist mutations (%) bb (A˚ ) 126 I (YR)(H)(TKE)(SQCDG) S(35) 119 D (R)(FWH)(K)(Y) Table 6. The top 25% of residues in 1sl8A at the interface with calcium Table 5. List of disruptive mutations for the top 25% of residues in 1sl8A, ion.(Field names: res: residue number in the PDB entry; type: amino acid that are at the interface with calcium ion. 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. )

Table 7. res type disruptive mutations 155 D (R)(FWH)(KYVCAG)(TQM) 160 G (KER)(FQMWHD)(NYLPI)(SVA) 157 D (R)(FWH)(Y)(VCAG) 166 E (FWH)(R)(Y)(VA) 159 S (R)(K)(FWH)(QM)

Table 7. List of disruptive mutations for the top 25% of residues in 1sl8A, that are at the interface with calcium ion.

Fig. 5. Residues in 1sl8A, at the interface with calcium ion, colored by their relative importance. The ligand (calcium 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 1sl8A.)

Figure 5 shows residues in 1sl8A colored by their importance, at the interface with 1sl8CA671. Calcium ion binding site. Table 6 lists the top 25% of residues at the interface with 1sl8CA669 (calcium ion). The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 3.6). Table 6. res type subst’s cvg noc/ dist (%) bb (A˚ ) Fig. 6. Residues in 1sl8A, at the interface with calcium ion, colored by their 155 D D(100) 0.07 6/2 2.28 relative importance. The ligand (calcium ion) is colored green. Atoms further 160 G G(100) 0.07 1/1 4.72 than 30A˚ away from the geometric center of the ligand, as well as on the line 157 D D(97) 0.08 7/3 2.24 of sight to the ligand were removed. (See Appendix for the coloring scheme N(2) for the protein chain 1sl8A.) 166 E E(97) 0.09 4/0 2.53 G(2) 159 S D(64) 0.20 5/3 2.48 Figure 6 shows residues in 1sl8A colored by their importance, at the continued in next column interface with 1sl8CA669. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1sl8A surface, away from (or

4 susbtantially larger than) other functional sites and interfaces reco- Table 9. continued gnizable in PDB entry 1sl8. It is shown in Fig. 7. The right panel res type disruptive shows (in blue) the rest of the larger cluster this surface belongs to. mutations

Table 9. Disruptive mutations for the surface patch in 1sl8A.

Another group of surface residues is shown in Fig.8. The residues

Fig. 7. A possible active surface on the chain 1sl8A. The larger cluster it belongs to is shown in blue.

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 22 F F(100) 0.07 31 G G(100) 0.07 37 E E(100) 0.07 26 D D(97)Q(2) 0.08 19 K K(94)Q(5) 0.10 21 M A(61)M(38) 0.13 14 W Q(58)W(38).(2) 0.17 Fig. 8. Another possible active surface on the chain 1sl8A. 25 L F(58)L(38)I(2) 0.17 28 N D(55)N(41)A(2) 0.21 belonging to this surface ”patch” are listed in Table 10, while Table 81 W F(76)W(23) 0.22 11 suggests possible disruptive replacements for these residues (see 17 R E(55)R(38).(2) 0.24 Section 3.6). A(2) Table 10. Table 8. Residues forming surface ”patch” in 1sl8A. res type substitutions(%) cvg 50 L L(100) 0.07 65 E E(100) 0.07 Table 9. 120 K K(100) 0.07 res type disruptive 124 G G(100) 0.07 mutations 155 D D(100) 0.07 22 F (KE)(TQD)(SNCRG)(M) 160 G G(100) 0.07 31 G (KER)(FQMWHD)(NYLPI)(SVA) 121 D D(97)N(2) 0.08 37 E (FWH)(YVCARG)(T)(SNKLPI) 157 D D(97)N(2) 0.08 26 D (FWHR)(Y)(VCAG)(T) 166 E E(97)G(2) 0.09 19 K (Y)(FTW)(SVCAG)(H) 59 R L(61)R(38) 0.13 21 M (Y)(H)(TR)(SCDG) 119 D D(97)G(2) 0.13 14 W (TE)(K)(D)(CG) 117 I V(58)I(38)M(2) 0.17 25 L (R)(Y)(T)(KE) 134 Y V(58)Y(38)M(2) 0.17 28 N (Y)(H)(FW)(R) 150 T M(58)T(38)L(2) 0.17 81 W (KE)(TQD)(SNCRG)(M) 151 F I(58)F(38)M(2) 0.17 17 R (T)(YD)(CG)(SVLAPI) 154 C A(58)C(38)G(2) 0.17 continued in next column continued in next column

5 Table 10. continued couple of residues should affect the protein somehow, with the exact res type substitutions(%) cvg effects to be determined experimentally. 60 H Q(58)H(38)N(2) 0.19 115 F F(97)L(2) 0.19 3.2 Known substitutions 159 S D(64)S(35) 0.20 One of the table columns is “substitutions” - other amino acid types 128 L A(55)L(41)P(2) 0.21 seen at the same position in the alignment. These amino acid types 66 A V(61)A(35)D(2) 0.23 may be interchangeable at that position in the protein, so if one wants 167 M F(58)M(41) 0.23 to affect the protein by a point mutation, they should be avoided. For 46 V V(82)I(17) 0.24 example if the substitutions are “RVK” and the original protein has 132 K R(58)K(38)I(2) 0.25 an R at that position, it is advisable to try anything, but RVK. Conver- 146 D E(58)D(38)Q(2) 0.25 sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with Table 10. Residues forming surface ”patch” in 1sl8A. 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 Table 11. guide - due to rounding errors these percentages often do not add up res type disruptive to 100%. mutations 3.3 Surface 50 L (YR)(TH)(SKECG)(FQWD) 65 E (FWH)(YVCARG)(T)(SNKLPI) To detect candidates for novel functional interfaces, first we look for 120 K (Y)(FTW)(SVCAG)(HD) residues that are solvent accessible (according to DSSP program) by ˚ 2 124 G (KER)(FQMWHD)(NYLPI)(SVA) at least 10A , which is roughly the area needed for one water mole- 155 D (R)(FWH)(KYVCAG)(TQM) cule to come in the contact with the residue. Furthermore, we require 160 G (KER)(FQMWHD)(NYLPI)(SVA) that these residues form a “cluster” of residues which have neighbor ˚ 121 D (R)(FWH)(Y)(VCAG) within 5A from any of their heavy atoms. 157 D (R)(FWH)(Y)(VCAG) Note, however, that, if our picture of protein evolution is correct, 166 E (FWH)(R)(Y)(VA) the neighboring residues which are not surface accessible might be 59 R (T)(YD)(SECG)(VA) equally important in maintaining the interaction specificity - they 119 D (R)(FWH)(K)(Y) should not be automatically dropped from consideration when choo- 117 I (Y)(R)(H)(T) sing the set for mutagenesis. (Especially if they form a cluster with 134 Y (K)(R)(Q)(E) the surface residues.) 150 T (R)(H)(K)(FW) 3.4 Number of contacts 151 F (T)(KE)(DR)(QCG) 154 C (KER)(QHD)(FMW)(Y) Another column worth noting is denoted “noc/bb”; it tells the num- 60 H (T)(E)(D)(SVCAG) ber of contacts heavy atoms of the residue in question make across 115 F (KE)(T)(QDR)(SCG) the interface, as well as how many of them are realized through the 159 S (R)(K)(FWH)(QM) backbone atoms (if all or most contacts are through the backbone, 128 L (YR)(H)(T)(KE) mutation presumably won’t have strong impact). Two heavy atoms ˚ 66 A (R)(KY)(EH)(Q) are considered to be “in contact” if their centers are closer than 5A. 167 M (TY)(SCHRG)(D)(KE) 3.5 Annotation 46 V (YR)(KE)(H)(QD) 132 K (Y)(T)(FW)(S) If the residue annotation is available (either from the pdb file or 146 D (FWHR)(Y)(VCAG)(T) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 11. Disruptive mutations for the surface patch in 1sl8A. 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- 3.1 Coverage 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

6 DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 9. COVERAGE 4.3 Credits

V 4.3.1 Alistat alistat reads a multiple sequence alignment from the 100% 50% 30% 5% 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 alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of V exact identities and len1, len2 are the unaligned lengths of the two RELATIVE IMPORTANCE 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 Fig. 9. Coloring scheme used to color residues by their relative importance. seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N numbers (hence, the most outlying sequence). alistat is copyrighted acid, and they are grouped in round brackets if they appear equally by HHMI/Washington University School of Medicine, 1992-2001, disruptive. From left to right, each bracketed group of amino acid and freely distributed under the GNU General Public License. types resembles more strongly the original (i.e. is, presumably, less 4.3.2 CE To map ligand binding sites from different disruptive) These suggestions are tentative - they might prove disrup- source structures, report maker uses the CE program: tive to the fold rather than to the interaction. Many researcher will http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) choose, however, the straightforward alanine mutations, especially in ”Protein structure alignment by incremental combinatorial extension the beginning stages of their investigation. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A˚ 2, 4.1 File formats which is roughly the area needed for one water molecule to come in Files with extension “ranks sorted” are the actual trace results. The the contact with the residue. DSSP is copyrighted by W. Kabsch, C. fields in the table in this file: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 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 4.3.4 HSSP Whenever available, report maker uses HSSP ali- • type amino acid type 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 http://swift.cmbi.kun.nl/swift/hssp/ 2. their type • rho ET score - the smaller this value, the lesser variability of 4.3.5 LaTex The text for this report was processed using LATEX; this position across the branches of the tree (and, presumably, Leslie Lamport, “LaTeX: A Document Preparation System Addison- the greater the importance for the protein) Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which 4.3.6 Muscle When making alignments “from scratch”, report have this rho or smaller maker uses Muscle alignment program: Edgar, Robert C. (2004), • gaps percentage of gaps in this column ”MUSCLE: multiple sequence alignment with high accuracy and 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 by cluster size: black is a single-residue cluster; clusters composed of 4.3.7 Pymol The figures in this report were produced using more than one residue colored according to this hierarchy (ordered Pymol. The scripts can be found in the attachment. Pymol by descending size): red, blue, yellow, green, purple, azure, tur- is an open-source application copyrighted by DeLano Scien- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tific LLC (2005). For more information about Pymol see bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, http://pymol.sourceforge.net/. (Note for Windows

7 users: the attached package needs to be unzipped for Pymol to read is copyrighted by Lichtarge Lab, Baylor College of Medicine, the scripts and launch the viewer.) Houston. 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 • 1sl8A.complex.pdb - coordinates of 1sl8A with all of its inter- visit: acting partners http://mammoth.bcm.tmc.edu/traceview/ • 1sl8A.etvx - ET viewer input file for 1sl8A The viewer is self-unpacking and self-installing. Input files to be used • 1sl8A.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 1sl8A main report. • 1sl8A.ranks - Ranks file in sequence order for 1sl8A 4.5 Citing this work • 1sl8A.clusters - Cluster descriptions for 1sl8A The method used to rank residues and make predictions in this report • 1sl8A.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 1sl8A Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1sl8A.descr - description of sequences used in 1sl8A msf by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1sl8A.ranks sorted - full listing of residues and their ranking for of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- 1sl8A tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. • 1sl8A.1sl8CA670.if.pml - Pymol script for Figure 4 report maker itself is described in Mihalek I., I. Res and O. • 1sl8A.cbcvg - used by other 1sl8A – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1sl8A.1sl8CA671.if.pml - Pymol script for Figure 5 of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 1sl8A.1sl8CA669.if.pml - Pymol script for Figure 6 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker

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