Pages 1–7 1w9c Evolutionary trace report by report maker June 18, 2010

4.3.1 Alistat 7 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 1w9c): Title: Proteolytic fragment of crm1 spanning six c-terminal heat repeats Compound: Mol id: 1; molecule: crm1 protein; chain: a, b; frag- ment: c-terminal six heat repeats, residues 707-1027; synonym: exportin 1; engineered: yes Organism, scientific name: Homo Sapiens; 1w9c contains a single unique chain 1w9cA (321 residues long) CONTENTS and its homologue 1w9cB.

1 Introduction 1 2 CHAIN 1W9CA 2.1 O14980 overview 2 Chain 1w9cA 1 2.1 O14980 overview 1 From SwissProt, id O14980, 100% identical to 1w9cA: 2.2 Multiple sequence alignment for 1w9cA 1 Description: Exportin-1 (Chromosome region maintenance 1 protein 2.3 Residue ranking in 1w9cA 2 homolog). 2.4 Top ranking residues in 1w9cA and their position on Organism, scientific name: Homo sapiens (Human). the structure 2 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.4.1 Clustering of residues at 25% coverage. 2 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4.2 Overlap with known functional surfaces at Catarrhini; Hominidae; Homo. 25% coverage. 2 Function: Mediates the nuclear export of cellular proteins (cargoes) 2.4.3 Possible novel functional surfaces at 25% bearing a leucine-rich nuclear export signal (NES) and of RNAs. In coverage. 3 the nucleus, in association with RANBP3, binds cooperatively to the NES on its target protein and to the GTPase Ran in its active GTP- 3 Notes on using trace results 5 bound form. Docking of this complex to the nuclear pore complex 3.1 Coverage 5 (NPC) is mediated through binding to nucleoporins. Upon transit 3.2 Known substitutions 6 of an nuclear export complex into the cytoplasm, disassembling of 3.3 Surface 6 the complex and hydrolysis of Ran-GTP to Ran-GDP (induced by 3.4 Number of contacts 6 RANBP1 and RANGAP1, respectively) cause release of the cargo 3.5 Annotation 6 from the export receptor. The directionality of nuclear export is 3.6 Mutation suggestions 6 thought to be conferred by an asymmetric distribution of the GTP- and GDP-bound forms of Ran between the cytoplasm and nucleus. 4 Appendix 6 Also mediates the nuclear export of the Rev protein of HIV-1 and 4.1 File formats 6 Rex protein of HTLV-1. Involved in Rex dimerization. Involved in 4.2 Color schemes used 6 U3 snoRNA transport from Cajal bodies to nucleoli. Binds to late 4.3 Credits 7 precursor U3 snoRNA bearing a TMG cap.

1 Lichtarge lab 2006 Subunit: Found in a nuclear export complex with RANBP3 and Ran. Found in a complex with HIV-1 Rev, HIV-1 Rev response ele- ment (RRE) RNA and Ran. Found in a complex with HTLV-1 Rex, RANBP3 and Ran. Interacts with DDX3X, NUP88, NUP214, HIV-1 Rev, HTLV-1 Rex and RANBP3. Subcellular location: Nuclear and cytoplasmic. Located in the nucleoplasm, Cajal bodies and nucleoli. Shuttles between the nucleus/nucleolus and the cytoplasm. Tissue specificity: Expressed in heart, brain, placenta, lung, liver, skeletal muscle, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes. Not expressed in the kidney. Fig. 1. Residues 707-866 in 1w9cA colored by their relative importance. (See Miscellaneous: Cellular target of leptomycin B (LMB), a Appendix, Fig.7, for the coloring scheme.) XPO1/CRM1 nuclear export inhibitor. Similarity: Belongs to the exportin family. Similarity: Contains 10 HEAT repeats. Similarity: Contains 1 importin N-terminal 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.

2.2 Multiple sequence alignment for 1w9cA Fig. 2. Residues 867-1027 in 1w9cA colored by their relative importance. For the chain 1w9cA, the alignment 1w9cA.msf (attached) with 71 (See Appendix, Fig.7, for the coloring scheme.) 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 2.4 Top ranking residues in 1w9cA and their position on to this report, under the name of 1w9cA.msf. Its statistics, from the the structure alistat program are the following: In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1w9cA colored Format: MSF by their importance: bright red and yellow indicate more conser- Number of sequences: 71 ved/important residues (see Appendix for the coloring scheme). A Total number of residues: 22519 Pymol script for producing this figure can be found in the attachment. Smallest: 281 Largest: 321 Average length: 317.2 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Alignment length: 321 top 25% of all residues, this time colored according to clusters they Average identity: 43% belong to. The clusters in Fig.4 are composed of the residues listed Most related pair: 98% in Table 1. Most unrelated pair: 19% Table 1. Most distant seq: 34% cluster size member color residues red 75 716,717,721,725,728,732,736 Furthermore, 2% of residues show as conserved in this alignment. 748,749,751,752,754,756,758 The alignment consists of 36% eukaryotic ( 4% vertebrata, 2% 784,785,787,788,795,796,797 arthropoda, 18% fungi, 1% plantae) sequences. (Descriptions of 798,799,800,801,822,823,827 some sequences were not readily available.) The file containing the 830,831,833,834,842,844,845 sequence descriptions can be found in the attachment, under the name 848,849,851,852,855,859,860 1w9cA.descr. 862,863,866,880,881,883,884 885,887,889,890,893,894,903 929,931,932,934,935,936,938 2.3 Residue ranking in 1w9cA 939,942,945,946,982,986,997 The 1w9cA sequence is shown in Figs. 1–2, with each residue colo- 1011,1016,1017,1018,1019 red according to its estimated importance. The full listing of residues continued in next column in 1w9cA can be found in the file called 1w9cA.ranks sorted in the attachment.

2 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. Interface with 1w9cB.Table 2 lists the top 25% of residues at the interface with 1w9cB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 709 Q Q(90) 0.07 15/13 3.19 E(5) I(1) .(2) 758 L L(85) 0.10 34/3 3.63 I(5) M(7) F(1) 754 E E(92) 0.12 45/7 2.61 D(2) S(1) N(1) Fig. 3. Residues in 1w9cA, colored by their relative importance. Clockwise: V(1) front, back, top and bottom views. 717 M V(4) 0.18 8/2 3.96 L(12) M(78) F(2) I(1) 748 M L(64) 0.21 11/5 3.62 M(29) V(4) Q(1) 751 V V(42) 0.21 16/4 3.54 I(46) M(2) A(5) S(2) 716 D D(84) 0.24 36/12 2.90 E(1) G(2) P(1) H(1) .(2) N(5)

Table 2. The top 25% of residues in 1w9cA at the interface with 1w9cB. (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 Fig. 4. Residues in 1w9cA, colored according to the cluster they belong to: in the bracket; noc/bb: number of contacts with the ligand, with the number of red, followed by blue and yellow are the largest clusters (see Appendix for contacts realized through backbone atoms given in the bracket; dist: distance the coloring scheme). Clockwise: front, back, top and bottom views. The of closest apporach to the ligand. ) corresponding Pymol script is attached.

Table 3. res type disruptive Table 1. continued mutations cluster size member 709 Q (Y)(H)(FTW)(CG) color residues continued in next column

Table 1. Clusters of top ranking residues in 1w9cA.

3 Table 3. continued res type disruptive mutations 758 L (YR)(T)(H)(SKECG) 754 E (H)(FW)(R)(Y) 717 M (Y)(T)(HR)(SCG) 748 M (Y)(H)(T)(R) 751 V (YR)(K)(E)(H) 716 D (R)(FWH)(Y)(VCAG)

Table 3. List of disruptive mutations for the top 25% of residues in 1w9cA, that are at the interface with 1w9cB. Fig. 6. A possible active surface on the chain 1w9cA. The larger cluster it belongs to is shown in blue.

Table 4. continued res type substitutions(%) cvg 845 R R(100) 0.02 880 W W(100) 0.02 884 H H(100) 0.02 894 L L(100) 0.02 788 Y Y(98)F(1) 0.03 860 F F(98)I(1) 0.03 736 G G(97)N(1)T(1) 0.04 844 H H(94)F(5) 0.04 848 F F(94)L(5) 0.04 935 H H(98)K(1) 0.04 787 D D(98)V(1) 0.05 827 F F(97)L(2) 0.06 752 K K(95)R(4) 0.07 800 V V(91)A(5)L(1) 0.07 I(1) 931 T T(91)C(2)M(5) 0.07 796 R R(87)K(12) 0.08 851 L L(92)F(5)M(1) 0.08 833 M M(95)I(4) 0.09 929 V V(88)T(2)A(7) 0.09 Fig. 5. Residues in 1w9cA, at the interface with 1w9cB, colored by their rela- I(1) tive importance. 1w9cB is shown in backbone representation (See Appendix 932 D D(92)Q(7) 0.09 for the coloring scheme for the protein chain 1w9cA.) 758 L L(85)I(5)M(7) 0.10 F(1) Figure 5 shows residues in 1w9cA colored by their importance, at the 938 G G(92)S(7) 0.10 interface with 1w9cB. 986 F F(90)Y(1)I(5) 0.10 S(1)R(1) 2.4.3 Possible novel functional surfaces at 25% coverage. One 831 L L(88)I(5)V(5) 0.11 group of residues is conserved on the 1w9cA surface, away from (or 1017 D D(97).(1)E(1) 0.11 susbtantially larger than) other functional sites and interfaces reco- 725 S S(94)G(1)F(1) 0.12 gnizable in PDB entry 1w9c. It is shown in Fig. 6. The right panel N(1)I(1) shows (in blue) the rest of the larger cluster this surface belongs to. 754 E E(92)D(2)S(1) 0.12 The residues belonging to this surface ”patch” are listed in Table N(1)V(1) 4, while Table 5 suggests possible disruptive replacements for these 887 R R(85)P(7)Y(4) 0.12 residues (see Section 3.6). T(1)F(1) Table 4. 939 L F(73)L(25)I(1) 0.13 res type substitutions(%) cvg 982 L L(91)F(7)I(1) 0.13 785 L L(100) 0.02 continued in next column 842 P P(100) 0.02 continued in next column

4 Table 4. continued Table 4. continued res type substitutions(%) cvg res type substitutions(%) cvg 1019 L L(95)I(1).(2) 0.13 H(2)V(1) 721 Y Y(92)F(5)C(1) 0.14 784 V I(21)V(76)L(2) 0.25 883 K R(15)Q(2)K(81) 0.14 823 F F(35)L(39)M(21) 0.25 903 N Y(2)R(2)N(88) 0.14 I(2)Y(1) Q(4)K(1) 749 R R(85)F(2)H(11) 0.15 Table 4. Residues forming surface ”patch” in 1w9cA. 795 A A(87)I(5)C(1) 0.15 S(1)T(2)V(1) 1018 F F(92)L(2)Y(2) 0.15 Table 5. .(1) res type disruptive 942 H H(36)Y(4)Q(57) 0.16 mutations E(1) 785 L (YR)(TH)(SKECG)(FQWD) 838 F F(85)M(8)L(4) 0.17 842 P (YR)(TH)(SKECG)(FQWD) T(1) 845 R (TD)(SYEVCLAPIG)(FMW)(N) 890 A A(50)E(38)M(5) 0.17 880 W (KE)(TQD)(SNCRG)(M) S(4)Q(1) 884 H (E)(TQMD)(SNKVCLAPIG)(YR) 1016 R R(87)A(5)H(2) 0.17 894 L (YR)(TH)(SKECG)(FQWD) K(2).(1) 788 Y (K)(Q)(EM)(NR) 717 M V(4)L(12)M(78) 0.18 860 F (KE)(T)(QDR)(SCG) F(2)I(1) 736 G (R)(KE)(FWH)(M) 728 I V(11)I(83)Q(5) 0.18 844 H (E)(TQD)(KM)(SNCG) 889 V V(73)T(5)I(18) 0.18 848 F (KE)(T)(QDR)(SCG) M(2) 935 H (TE)(D)(SVMCAG)(QLPI) 732 I I(39)V(57).(1) 0.19 787 D (R)(H)(FKYW)(QCG) S(1) 827 F (KE)(T)(QDR)(SCG) 866 I L(63)M(4)I(26) 0.19 752 K (Y)(T)(FW)(SVCAG) Y(5) 800 V (YR)(KE)(H)(QD) 936 T K(66)V(4)A(5) 0.19 931 T (R)(K)(H)(FW) T(21)L(1)I(1) 796 R (T)(YD)(SVCAG)(FELWPI) 797 E D(43)E(56) 0.20 851 L (YR)(T)(H)(KECG) 849 F F(64)Y(33)I(1) 0.20 833 M (Y)(TH)(R)(SCG) 1024 E Q(5)E(84)V(5) 0.20 929 V (R)(K)(YE)(H) M(1).(2) 932 D (FWHR)(Y)(VCAG)(T) 748 M L(64)M(29)V(4) 0.21 758 L (YR)(T)(H)(SKECG) Q(1) 938 G (KR)(E)(FQMWH)(D) 751 V V(42)I(46)M(2) 0.21 986 F (K)(E)(Q)(D) A(5)S(2) 831 L (YR)(H)(T)(KE) 863 F L(67)F(29)I(2) 0.21 1017 D (R)(FW)(H)(VCAG) 799 E E(77)K(8)G(5) 0.22 725 S (R)(K)(H)(EQ) Q(2)L(4)T(1) 754 E (H)(FW)(R)(Y) 885 T T(52)D(22)E(7) 0.22 887 R (D)(E)(T)(S) H(1)N(14)V(1) 939 L (R)(Y)(T)(KE) P(1) 982 L (R)(Y)(T)(KE) 934 S F(18)S(22)D(46) 0.23 1019 L (Y)(R)(T)(H) L(9)A(1)N(1) 721 Y (K)(Q)(M)(E) 997 F L(2)I(5)F(84) 0.23 883 K (Y)(T)(FW)(SVCAG) H(1)T(2).(1) 903 N (Y)(T)(FW)(VA) C(1) 749 R (TD)(E)(SCG)(VLAPI) 716 D D(84)E(1)G(2) 0.24 795 A (R)(K)(E)(Y) P(1)H(1).(2) 1018 F (K)(E)(Q)(TD) N(5) 942 H (TE)(VMA)(QCDG)(SKLPI) 798 P A(52)P(32)S(11) 0.24 continued in next column continued in next column

5 Table 5. continued 3.3 Surface res type disruptive To detect candidates for novel functional interfaces, first we look for mutations residues that are solvent accessible (according to DSSP program) by 838 F (K)(E)(R)(TQD) 2 at least 10A˚ , which is roughly the area needed for one water mole- 890 A (Y)(R)(H)(K) cule to come in the contact with the residue. Furthermore, we require 1016 R (TD)(YE)(S)(CG) that these residues form a “cluster” of residues which have neighbor 717 M (Y)(T)(HR)(SCG) within 5A˚ from any of their heavy atoms. 728 I (Y)(R)(H)(T) Note, however, that, if our picture of protein evolution is correct, 889 V (R)(Y)(K)(EH) the neighboring residues which are not surface accessible might be 732 I (R)(Y)(H)(K) equally important in maintaining the interaction specificity - they 866 I (R)(Y)(TH)(K) should not be automatically dropped from consideration when choo- 936 T (R)(K)(H)(FW) sing the set for mutagenesis. (Especially if they form a cluster with 797 E (FWH)(R)(YVCAG)(T) the surface residues.) 849 F (K)(E)(Q)(R) 1024 E (H)(FW)(Y)(CG) 3.4 Number of contacts 748 M (Y)(H)(T)(R) Another column worth noting is denoted “noc/bb”; it tells the num- 751 V (YR)(K)(E)(H) ber of contacts heavy atoms of the residue in question make across 863 F (KE)(T)(R)(QD) the interface, as well as how many of them are realized through the 799 E (FWH)(Y)(R)(VA) backbone atoms (if all or most contacts are through the backbone, 885 T (R)(K)(H)(FW) mutation presumably won’t have strong impact). Two heavy atoms 934 S (R)(K)(H)(YQ) are considered to be “in contact” if their centers are closer than 5A˚ . 997 F (K)(E)(Q)(D) 716 D (R)(FWH)(Y)(VCAG) 3.5 Annotation 798 P (R)(Y)(E)(K) If the residue annotation is available (either from the pdb file or 784 V (YR)(KE)(H)(QD) from other sources), another column, with the header “annotation” 823 F (K)(E)(T)(R) appears. Annotations carried over from PDB are the following: site (indicating existence of related site record in PDB ), S-S (disulfide Table 5. Disruptive mutations for the surface patch in 1w9cA. 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 disruptive) These suggestions are tentative - they might prove disrup- 3.2 Known substitutions tive to the fold rather than to the interaction. Many researcher will One of the table columns is “substitutions” - other amino acid types choose, however, the straightforward alanine mutations, especially in seen at the same position in the alignment. These amino acid types the beginning stages of their investigation. 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 4 APPENDIX example if the substitutions are “RVK” and the original protein has 4.1 File formats 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, Files with extension “ranks sorted” are the actual trace results. The one may try replacing, R with K, or (perhaps more surprisingly), with fields in the table in this file: V. The percentage of times the substitution appears in the alignment • alignment# number of the position in the alignment is given in the immediately following bracket. No percentage is given • residue# residue number in the PDB file 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 type amino acid type to 100%. • rank rank of the position according to older version of ET

6 by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License. 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: COVERAGE http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension V (CE) of the optimal path . Protein Engineering 11(9) 739-747. 100% 50% 30% 5% 4.3.3 DSSP In this work a residue is considered solvent accessi- ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version V by [email protected] November 18,2002, RELATIVE IMPORTANCE http://www.cmbi.kun.nl/gv/dssp/descrip.html. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- Fig. 7. Coloring scheme used to color residues by their relative importance. gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- • variability has two subfields: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 1. number of different amino acids appearing in in this column of the alignment http://swift.cmbi.kun.nl/swift/hssp/ 2. their type 4.3.5 LaTex The text for this report was processed using LATEX; • rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”MUSCLE: multiple sequence alignment with high accuracy and • gaps percentage of gaps in this column high throughput.” Nucleic Acids Research 32(5), 1792-97. 4.2 Color schemes used http://www.drive5.com/muscle/ The following color scheme is used in figures with residues colored 4.3.7 Pymol The figures in this report were produced using by cluster size: black is a single-residue cluster; clusters composed of Pymol. The scripts can be found in the attachment. Pymol more than one residue colored according to this hierarchy (ordered is an open-source application copyrighted by DeLano Scien- by descending size): red, blue, yellow, green, purple, azure, tur- tific LLC (2005). For more information about Pymol see quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, http://pymol.sourceforge.net/. (Note for Windows bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, users: the attached package needs to be unzipped for Pymol to read DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, the scripts and launch the viewer.) tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated 4.4 Note about ET Viewer evolutionary pressure they experience can be seen in Fig. 7. Dan Morgan from the Lichtarge lab has developed a visualization tool specifically for viewing trace results. If you are interested, please 4.3 Credits visit: 4.3.1 Alistat alistat reads a multiple sequence alignment from the http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number The viewer is self-unpacking and self-installing. Input files to be used of residues, the average and range of the sequence lengths, and the with ETV (extension .etvx) can be found in the attachment to the alignment length (e.g. including gap characters). Also shown are main report. some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of 4.5 Citing this work exact identities and len1, len2 are the unaligned lengths of the two The method used to rank residues and make predictions in this report sequences. The ”average percent identity”, ”most related pair”, and can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ”most unrelated pair” of the alignment are the average, maximum, Evolution-Entropy Hybrid Methods for Ranking of Protein Residues and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant by Importance” J. Mol. Bio. 336: 1265-82. For the original version seq” is calculated by finding the maximum pairwise identity (best of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- relative) for all N sequences, then finding the minimum of these N tionary Trace Method Defines Binding Surfaces Common to Protein numbers (hence, the most outlying sequence). alistat is copyrighted Families” J. Mol. Bio. 257: 342-358.

7 report maker itself is described in Mihalek I., I. Res and O. • 1w9cA.etvx - ET viewer input file for 1w9cA Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1w9cA.cluster report.summary - Cluster report summary for of service for comparative analysis of proteins.” Bioinformatics 1w9cA 22:1656-7. • 1w9cA.ranks - Ranks file in sequence order for 1w9cA 4.6 About report maker • 1w9cA.clusters - Cluster descriptions for 1w9cA report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 1w9cA.msf - the multiple sequence alignment used for the chain king visualization program was written by Ivica Res.ˇ report maker 1w9cA is copyrighted by Lichtarge Lab, Baylor College of Medicine, • 1w9cA.descr - description of sequences used in 1w9cA msf Houston. • 1w9cA.ranks sorted - full listing of residues and their ranking 4.7 Attachments for 1w9cA The following files should accompany this report: • 1w9cA.1w9cB.if.pml - Pymol script for Figure 5 • 1w9cA.cbcvg - used by other 1w9cA – related pymol scripts • 1w9cA.complex.pdb - coordinates of 1w9cA with all of its interacting partners

8