Pages 1–6 2i0v Evolutionary trace report by report maker September 17, 2008

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

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 2i0v): Title: C-fms tyrosine kinase in complex with a quinolone inhibitor Compound: Mol id: 1; molecule: cfms tyrosine kinase; chain: a; fragment: kinase domain; ec: 2.7.10.1; engineered: yes Organism, scientific name: Homo Sapiens; CONTENTS 2i0v contains a single unique chain 2i0vA (303 residues long).

1 Introduction 1

2 Chain 2i0vA 1 2.1 Q4SQ12 overview 1 2 CHAIN 2I0VA 2.2 Multiple sequence alignment for 2i0vA 1 2.1 Q4SQ12 overview 2.3 Residue ranking in 2i0vA 1 From SwissProt, id Q4SQ12, 63% identical to 2i0vA: 2.4 Top ranking residues in 2i0vA and their position on Description: Chromosome 7 SCAF14536, whole genome shotgun the structure 1 sequence. (Fragment). 2.4.1 Clustering of residues at 25% coverage. 2 Organism, scientific name: Tetraodon nigroviridis (Green puffer). 2.4.2 Overlap with known functional surfaces at : Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 25% coverage. 2 Euteleostomi; ; ; Teleostei; Euteleo- 2.4.3 Possible novel functional surfaces at 25% stei; ; ; Acanthopterygii; ; coverage. 3 ; Tetradontoidea; ; Tetraodon. Caution: 3 Notes on using trace results 4 The sequence shown here is derived from an EMBL/GenBank/DDBJ whole genome shotgun (WGS) entry 3.1 Coverage 4 which is preliminary data. 3.2 Known substitutions 5 3.3 Surface 5 3.4 Number of contacts 5 2.2 Multiple sequence alignment for 2i0vA 3.5 Annotation 5 3.6 Mutation suggestions 5 For the chain 2i0vA, the alignment 2i0vA.msf (attached) with 39 sequences was used. The alignment was assembled through combi- 4 Appendix 5 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 5 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 5 report, under the name of 2i0vA.msf. Its statistics, from the alistat 4.3 Credits 6 program are the following:

1 Lichtarge lab 2006 residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 544-763 in 2i0vA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 764-916 in 2i0vA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Fig. 3. Residues in 2i0vA, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 39 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 11319 top 25% of all residues, this time colored according to clusters they Smallest: 231 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 303 in Table 1. Average length: 290.2 Alignment length: 303 Table 1. Average identity: 54% cluster size member Most related pair: 99% color residues Most unrelated pair: 36% red 74 596,614,615,616,617,618,619 Most distant seq: 43% 630,633,643,645,649,650,651 664,669,671,675,679,761,765 766,769,775,776,777,778,780 Furthermore, 11% of residues show as conserved in this alignment. 781,782,783,785,793,794,796 The alignment consists of 94% eukaryotic ( 56% vertebrata, 797,798,799,800,801,809,817 20% arthropoda), and 2% viral sequences. (Descriptions of some 818,820,821,822,823,824,825 sequences were not readily available.) The file containing the 827,836,837,838,839,842,844 sequence descriptions can be found in the attachment, under the name 846,847,848,851,852,855,856 2i0vA.descr. 857,872,874,878,886,889,892 893,900,901,903 2.3 Residue ranking in 2i0vA blue 2 653,658 The 2i0vA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues Table 1. Clusters of top ranking residues in 2i0vA. in 2i0vA can be found in the file called 2i0vA.ranks sorted in the attachment. 2.4.2 Overlap with known functional surfaces at 25% coverage. 2.4 Top ranking residues in 2i0vA and their position on The name of the ligand is composed of the source PDB identifier the structure and the heteroatom name used in that file. In the following we consider residues ranking among top 25% of resi- 6C3 binding site. Table 2 lists the top 25% of residues at the inter- dues in the protein . Figure 3 shows residues in 2i0vA colored by their face with 2i0v6C31000 (6c3). The following table (Table 3) suggests importance: bright red and yellow indicate more conserved/important possible disruptive replacements for these residues (see Section 3.6).

2 Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 614 A A(100) 0.11 10/3 3.51 616 K K(100) 0.11 9/0 3.16 664 E E(100) 0.11 8/8 3.36 669 G G(100) 0.11 9/9 3.75 785 L L(100) 0.11 30/0 3.40 796 D D(100) 0.11 3/3 3.62 797 F F(100) 0.11 37/5 3.75 801 R R(100) 0.11 9/0 3.90 596 V V(94) 0.18 17/0 3.98 .(5) 800 A A(82) 0.20 16/8 3.17 T(7) S(10)

Table 2. The top 25% of residues in 2i0vA at the interface with 6C3.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: Fig. 4. Residues in 2i0vA, colored according to the cluster they belong to: distance of closest apporach to the ligand. ) 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. Table 3. res type disruptive mutations 614 A (KYER)(QHD)(N)(FTMW) 616 K (Y)(FTW)(SVCAG)(HD) 664 E (FWH)(YVCARG)(T)(SNKLPI) 669 G (KER)(FQMWHD)(NYLPI)(SVA) 785 L (YR)(TH)(SKECG)(FQWD) 796 D (R)(FWH)(KYVCAG)(TQM) 797 F (KE)(TQD)(SNCRG)(M) 801 R (TD)(SYEVCLAPIG)(FMW)(N) 596 V (KYER)(QHD)(N)(FTMW) 800 A (KR)(E)(Y)(QH)

Table 3. List of disruptive mutations for the top 25% of residues in 2i0vA, that are at the interface with 6C3.

Figure 5 shows residues in 2i0vA colored by their importance, at the interface with 2i0v6C31000. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2i0vA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2i0v. It is shown in Fig. 6. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type substitutions(%) cvg 614 A A(100) 0.11 616 K K(100) 0.11 continued in next column

3 Table 4. continued res type substitutions(%) cvg 798 G G(100) 0.11 801 R R(100) 0.11 809 Y Y(100) 0.11 821 W W(100) 0.11 825 E E(100) 0.11 836 S S(100) 0.11 837 D D(100) 0.11 839 W W(100) 0.11 846 W W(100) 0.11 847 E E(100) 0.11 649 L L(97)F(2) 0.12 822 M M(97)T(2) 0.12 761 Q Q(92)D(7) 0.14 799 L L(94)F(5) 0.15 878 P P(97).(2) 0.16 893 W W(97).(2) 0.16 619 K K(94)R(2)Q(2) 0.17 857 P P(89)S(2)C(7) 0.17 872 G G(94)A(2)D(2) 0.17 596 V V(94).(5) 0.18 Fig. 5. Residues in 2i0vA, at the interface with 6C3, colored by their relative 650 L L(94)I(5) 0.18 importance. The ligand (6C3) is colored green. Atoms further than 30A˚ away 679 R R(92)N(5)K(2) 0.18 from the geometric center of the ligand, as well as on the line of sight to the 617 M M(84)T(7)R(7) 0.19 ligand were removed. (See Appendix for the coloring scheme for the protein 630 L L(82)F(15)I(2) 0.20 chain 2i0vA.) 800 A A(82)T(7)S(10) 0.20 886 Y Y(94)H(2).(2) 0.20 900 R R(94)K(2).(2) 0.21 901 P P(94)Q(2).(2) 0.21 903 F F(94)Q(2).(2) 0.21 817 L L(89)V(5)M(2) 0.22 F(2) 824 P P(84)I(10)T(5) 0.22 851 L Y(7)L(89)F(2) 0.23 856 Y Y(94)N(5) 0.23 820 K K(82)R(17) 0.24 827 I L(76)I(23) 0.24 618 L V(5)L(84)M(10) 0.25 Fig. 6. A possible active surface on the chain 2i0vA. The larger cluster it 641 G G(84)E(5).(7) 0.25 belongs to is shown in blue. D(2) 874 Q R(92)Q(5).(2) 0.25

Table 4. continued Table 4. Residues forming surface ”patch” in 2i0vA. res type substitutions(%) cvg 633 E E(100) 0.11 643 H H(100) 0.11 Table 5. 645 N N(100) 0.11 res type disruptive 664 E E(100) 0.11 mutations 669 G G(100) 0.11 614 A (KYER)(QHD)(N)(FTMW) 777 R R(100) 0.11 616 K (Y)(FTW)(SVCAG)(HD) 778 D D(100) 0.11 633 E (FWH)(YVCARG)(T)(SNKLPI) 782 R R(100) 0.11 643 H (E)(TQMD)(SNKVCLAPIG)(YR) 785 L L(100) 0.11 645 N (Y)(FTWH)(SEVCARG)(MD) 793 K K(100) 0.11 664 E (FWH)(YVCARG)(T)(SNKLPI) 796 D D(100) 0.11 669 G (KER)(FQMWHD)(NYLPI)(SVA) 797 F F(100) 0.11 continued in next column continued in next column

4 Table 5. continued in a chain], according to trace. The ET results are presented in the res type disruptive form of a table, usually limited to top 25% percent of residues (or mutations to some nearby percentage), sorted by the strength of the presumed 777 R (TD)(SYEVCLAPIG)(FMW)(N) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 778 D (R)(FWH)(KYVCAG)(TQM) pressure on the residue.) Starting from the top of that list, mutating a 782 R (TD)(SYEVCLAPIG)(FMW)(N) couple of residues should affect the protein somehow, with the exact 785 L (YR)(TH)(SKECG)(FQWD) effects to be determined experimentally. 793 K (Y)(FTW)(SVCAG)(HD) 796 D (R)(FWH)(KYVCAG)(TQM) 3.2 Known substitutions 797 F (KE)(TQD)(SNCRG)(M) One of the table columns is “substitutions” - other amino acid types 798 G (KER)(FQMWHD)(NYLPI)(SVA) seen at the same position in the alignment. These amino acid types 801 R (TD)(SYEVCLAPIG)(FMW)(N) may be interchangeable at that position in the protein, so if one wants 809 Y (K)(QM)(NEVLAPIR)(D) to affect the protein by a point mutation, they should be avoided. For 821 W (KE)(TQD)(SNCRG)(M) example if the substitutions are “RVK” and the original protein has 825 E (FWH)(YVCARG)(T)(SNKLPI) an R at that position, it is advisable to try anything, but RVK. Conver- 836 S (KR)(FQMWH)(NYELPI)(D) sely, when looking for substitutions which will not affect the protein, 837 D (R)(FWH)(KYVCAG)(TQM) one may try replacing, R with K, or (perhaps more surprisingly), with 839 W (KE)(TQD)(SNCRG)(M) V. The percentage of times the substitution appears in the alignment 846 W (KE)(TQD)(SNCRG)(M) is given in the immediately following bracket. No percentage is given 847 E (FWH)(YVCARG)(T)(SNKLPI) in the cases when it is smaller than 1%. This is meant to be a rough 649 L (R)(TY)(KE)(SCHG) guide - due to rounding errors these percentages often do not add up 822 M (YH)(R)(FTW)(SKCDG) to 100%. 761 Q (Y)(FWH)(T)(VCAG) 799 L (R)(TY)(KE)(SCHG) 3.3 Surface 878 P (YR)(TH)(SCG)(KE) To detect candidates for novel functional interfaces, first we look for 893 W (KE)(TQD)(SNCG)(R) residues that are solvent accessible (according to DSSP program) by 2 619 K (Y)(T)(FW)(SVCAG) at least 10A˚ , which is roughly the area needed for one water mole- 857 P (R)(Y)(H)(K) cule to come in the contact with the residue. Furthermore, we require 872 G (R)(K)(H)(E) that these residues form a “cluster” of residues which have neighbor 596 V (KYER)(QHD)(N)(FTMW) within 5A˚ from any of their heavy atoms. 650 L (YR)(TH)(SKECG)(FQWD) Note, however, that, if our picture of protein evolution is correct, 679 R (T)(Y)(D)(SVCAG) the neighboring residues which are not surface accessible might be 617 M (Y)(H)(T)(CG) equally important in maintaining the interaction specificity - they 630 L (R)(Y)(T)(KE) should not be automatically dropped from consideration when choo- 800 A (KR)(E)(Y)(QH) sing the set for mutagenesis. (Especially if they form a cluster with 886 Y (K)(M)(Q)(EVLAPI) the surface residues.) 900 R (T)(D)(Y)(VCAG) 901 P (Y)(R)(TH)(CG) 3.4 Number of contacts 903 F (TE)(K)(D)(CG) Another column worth noting is denoted “noc/bb”; it tells the num- 817 L (YR)(T)(H)(KE) ber of contacts heavy atoms of the residue in question make across 824 P (R)(Y)(H)(K) the interface, as well as how many of them are realized through the 851 L (R)(K)(TE)(Y) backbone atoms (if all or most contacts are through the backbone, 856 Y (K)(M)(EVQAR)(LPI) mutation presumably won’t have strong impact). Two heavy atoms 820 K (Y)(T)(FW)(SVCAG) are considered to be “in contact” if their centers are closer than 5A˚ . 827 I (YR)(TH)(SKECG)(FQWD) 618 L (Y)(R)(H)(T) 3.5 Annotation 641 G (R)(FKWH)(QM)(YE) If the residue annotation is available (either from the pdb file or 874 Q (Y)(T)(FW)(SVCAHG) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 5. Disruptive mutations for the surface patch in 2i0vA. (indicating existence of related site record in PDB ), S-S (disulfide 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 GST C], medium [LP NQDEMIK], large

5 more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, COVERAGE tan, DarkOrange, DeepPink, maroon, BlanchedAlmond.

V The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 7. 100% 50% 30% 5% 4.3 Credits 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number V of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are RELATIVE IMPORTANCE some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of Fig. 7. Coloring scheme used to color residues by their relative importance. exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant tively [KHR], or negatively [DE] charged, aromatic [W F Y H], seq” is calculated by finding the maximum pairwise identity (best long aliphatic chain [EKRQM], OH-group possession [SDET Y ], relative) for all N sequences, then finding the minimum of these N and NH2 group possession [NQRK]. The suggestions are listed numbers (hence, the most outlying sequence). alistat is copyrighted according to how different they appear to be from the original amino by HHMI/Washington University School of Medicine, 1992-2001, acid, and they are grouped in round brackets if they appear equally and freely distributed under the GNU General Public License. disruptive. From left to right, each bracketed group of amino acid 4.3.2 CE To map ligand binding sites from different types resembles more strongly the original (i.e. is, presumably, less source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A˚ , which is roughly the area needed for one water molecule to come in 4.1 File formats the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Files with extension “ranks sorted” are the actual trace results. The Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version fields in the table in this file: by [email protected] November 18,2002,

• alignment# number of the position in the alignment http://www.cmbi.kun.nl/gv/dssp/descrip.html. • residue# residue number in the PDB file 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 75% of the query are taken out, however); R. Schneider, A. de • rank rank of the position according to older version of ET 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

6 is an open-source application copyrighted by DeLano Scien- 4.6 About report maker tific LLC (2005). For more information about Pymol see report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://pymol.sourceforge.net/. (Note for Windows king visualization program was written by Ivica Res.ˇ report maker 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 • visit: 2i0vA.complex.pdb - coordinates of 2i0vA with all of its inter- acting partners http://mammoth.bcm.tmc.edu/traceview/ • 2i0vA.etvx - ET viewer input file for 2i0vA The viewer is self-unpacking and self-installing. Input files to be used • 2i0vA.cluster report.summary - Cluster report summary for with ETV (extension .etvx) can be found in the attachment to the 2i0vA main report. • 2i0vA.ranks - Ranks file in sequence order for 2i0vA 4.5 Citing this work • 2i0vA.clusters - Cluster descriptions for 2i0vA The method used to rank residues and make predictions in this report • 2i0vA.msf - the multiple sequence alignment used for the chain can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 2i0vA Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 2i0vA.descr - description of sequences used in 2i0vA msf 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- 2i0vA.ranks sorted - full listing of residues and their ranking for tionary Trace Method Defines Binding Surfaces Common to Protein 2i0vA Families” J. Mol. Bio. 257: 342-358. • 2i0vA.2i0v6C31000.if.pml - Pymol script for Figure 5 report maker itself is described in Mihalek I., I. Res and O. • 2i0vA.cbcvg - used by other 2i0vA – related pymol scripts Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type of service for comparative analysis of proteins.” Bioinformatics 22:1656-7.

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