Pages 1–7 1cts Evolutionary trace report by report maker July 3, 2009

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 7 4.5 Citing this work 7 4.6 About report maker 7 4.7 Attachments 7

1 INTRODUCTION From the original Data Bank entry (PDB id 1cts): Title: Crystallographic refinement and atomic models of two diffe- rent forms of at 2.7 and 1.7 angstroms resolution Compound: Mol id: 1; molecule: citrate synthase; chain: a; ec: 4.1.3.7; engineered: yes Organism, scientific name: Sus Scrofa 1cts contains a single unique chain 1ctsA (437 residues long).

2 CHAIN 1CTSA 2.1 P00889 overview CONTENTS From SwissProt, id P00889, 100% identical to 1ctsA: 1 Introduction 1 Description: Citrate synthase, mitochondrial precursor (EC 2.3.3.1). Organism, scientific name: Sus scrofa (Pig). 2 Chain 1ctsA 1 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.1 P00889 overview 1 Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; 2.2 Multiple sequence alignment for 1ctsA 1 Suina; Suidae; Sus. 2.3 Residue ranking in 1ctsA 1 Catalytic activity: Acetyl-CoA + H(2)O + oxaloacetate = citrate + 2.4 Top ranking residues in 1ctsA and their position on CoA. the structure 1 Pathway: Tricarboxylic acid cycle. 2.4.1 Clustering of residues at 25% coverage. 1 Subunit: Homodimer. 2.4.2 Overlap with known functional surfaces at Subcellular location: Mitochondrial matrix. 25% coverage. 2 Miscellaneous: Citrate synthase is found in nearly all cells capable of oxidative metabolism. 3 Notes on using trace results 5 Similarity: Belongs to the citrate synthase family. 3.1 Coverage 5 About: This Swiss-Prot entry is copyright. It is produced through a 3.2 Known substitutions 5 collaboration between the Swiss Institute of Bioinformatics and the 3.3 Surface 5 EMBL outstation - the European Bioinformatics Institute. There are 3.4 Number of contacts 5 no restrictions on its use as long as its content is in no way modified 3.5 Annotation 5 and this statement is not removed. 3.6 Mutation suggestions 5 2.2 Multiple sequence alignment for 1ctsA 4 Appendix 6 For the chain 1ctsA, the alignment 1ctsA.msf (attached) with 193 4.1 File formats 6 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 6 database, and fragments shorter than 75% of the query as well as 4.3 Credits 6 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 6 to this report, under the name of 1ctsA.msf. Its statistics, from the 4.3.2 CE 6 alistat program are the following:

1 Lichtarge lab 2006 2.4 Top ranking residues in 1ctsA and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1ctsA 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.

Fig. 1. Residues 1-218 in 1ctsA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 219-437 in 1ctsA colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) Fig. 3. Residues in 1ctsA, colored by their relative importance. Clockwise: front, back, top and bottom views.

Format: MSF Number of sequences: 193 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Total number of residues: 80831 top 25% of all residues, this time colored according to clusters they Smallest: 329 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 437 in Table 1. Average length: 418.8 Table 1. Alignment length: 437 Average identity: 48% cluster size member Most related pair: 99% color residues Most unrelated pair: 22% red 98 56,58,59,63,64,66,71,89,90 Most distant seq: 38% 94,96,132,136,137,138,140 152,154,174,178,180,189,229 231,235,237,238,239,240,241 Furthermore, 1% of residues show as conserved in this alignment. 242,243,244,245,246,249,250 The alignment consists of 30% eukaryotic ( 2% vertebrata, 1% 251,252,253,254,255,256,257 arthropoda, 15% fungi, 5% plantae), and 7% prokaryotic sequences. 260,264,265,268,269,270,271 (Descriptions of some sequences were not readily available.) The file 272,273,274,275,276,277,278 containing the sequence descriptions can be found in the attachment, 279,281,282,284,314,316,317 under the name 1ctsA.descr. 318,319,320,321,322,323,324 327,328,329,330,370,372,373 2.3 Residue ranking in 1ctsA 374,375,378,379,382,386,393 394,395,397,398,400,401,404 The 1ctsA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 1ctsA can be found in the file called 1ctsA.ranks sorted in the attachment.

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

Table 2. The top 25% of residues in 1ctsA at the interface with .(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: distance of closest apporach to the ligand. )

Table 3. res type disruptive mutations 238 H (E)(TQMD)(SNKVCLAPIG)(YR) 274 H (TEQM)(KVCAG)(SNLPDI)(R) 242 N (Y)(FWH)(TER)(SVA) 273 L (T)(Y)(ER)(CG) 329 R (TD)(SVCLAPIG)(YE)(FMW) 397 F (K)(E)(Q)(D) 401 R (T)(D)(CG)(Y) Fig. 4. Residues in 1ctsA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for Table 3. List of disruptive mutations for the top 25% of residues in 1ctsA, the coloring scheme). Clockwise: front, back, top and bottom views. The that are at the interface with citric acid. corresponding Pymol script is attached.

Table 1. continued cluster size member color residues 420,421,422,423,424 blue 3 78,79,102

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

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. Citric acid . Table 2 lists the top 25% of residues at the interface with 1ctsCIT1 (citric acid). 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˚ ) 238 H H(100) 0.01 37/5 2.62 274 H H(99)D 0.02 29/6 3.26 242 N N(98).C 0.03 25/4 3.36 273 L L(98)RF 0.03 3/3 4.08 Fig. 5. Residues in 1ctsA, at the interface with citric acid, colored by their 329 R R(99). 0.04 2/0 4.49 relative importance. The ligand (citric acid) is colored green. Atoms further 397 F F(98) 0.09 5/0 4.59 than 30A˚ away from the geometric center of the ligand, as well as on the line .(1)Y of sight to the ligand were removed. (See Appendix for the coloring scheme 401 R R(98) 0.09 14/0 2.69 for the protein chain 1ctsA.) .(1)M continued in next column Figure 5 shows residues in 1ctsA colored by their importance, at the interface with 1ctsCIT1.

3 Interface with 1ctsA1.Table 4 lists the top 25% of residues at Table 4. continued the interface with 1ctsA1. The following table (Table 5) suggests res type subst’s cvg noc/ dist possible disruptive replacements for these residues (see Section 3.6). (%) bb (A˚ ) .(2)RAT Table 4. 254 A S(3) 0.15 52/38 2.92 res type subst’s cvg noc/ dist A(83) (%) bb (A˚ ) T(11) 271 G G(100) 0.01 22/22 3.01 G(1)P 274 H H(99)D 0.02 3/2 4.30 255 L N(1) 0.16 38/20 3.15 242 N N(98).C 0.03 2/2 4.97 L(93) 273 L L(98)RF 0.03 53/12 3.15 Q(3)DRV 138 M M(99)V 0.04 1/1 4.52 H 272 P P(98)RE 0.04 48/11 2.92 264 A A(90) 0.17 20/13 3.77 246 H S(5) 0.06 38/3 3.31 G(5) H(86)L S(4) F(7)A 44 G G(90) 0.18 39/39 2.85 268 G A(17) 0.06 17/17 3.48 .(8) G(82)T N(1) 239 E E(97)G. 0.07 24/4 2.58 270 A FA(82) 0.19 4/3 4.09 T(1)D K(6) 241 G Q(5) 0.08 25/25 3.13 S(4) G(86)P W(4) L(6)IFN Y(1) 243 V C(5) 0.08 22/3 3.30 260 L A(4) 0.20 36/13 2.91 V(82) L(75) A(11)LS S(7) 136 H H(97).S 0.09 1/0 4.71 Y(8) DI T(1) 253 S S(93) 0.09 5/4 3.78 F(1)IG A(5) 256 S A(10) 0.21 24/8 2.82 T(1) S(83) 240 G .(11) 0.10 7/7 3.26 G(3)ETV G(76)V . S(10)M 424 S Q(5) 0.22 51/19 3.26 250 L L(89) 0.11 38/3 3.31 S(83) V(7)A .(2) M(1)QI A(6)NTY 251 V V(89) 0.11 28/8 3.15 DG I(3) 58 L I(6) 0.23 2/0 4.54 C(5)TA L(87) 56 S T(7) 0.12 2/2 4.60 .(1) S(90) V(4) .(1)H C(1) 132 P P(95)A. 0.13 21/3 3.05 420 E G(4) 0.24 37/13 3.17 D(1)SRL E(84) 276 L G(13) 0.13 1/0 4.78 .(2) L(74) I(6)VRK R(10) CNY M(1)K 47 G N(9) 0.25 6/6 4.37 421 R R(96) 0.14 28/19 2.85 S(1) .(2) G(75) P(1)Y .(1) 422 P P(96) 0.14 59/29 3.13 D(9) continued in next column A(1)T 423 K R(11) 0.25 61/33 2.58 continued in next column

4 Table 4. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) K(80) G(4) .(2)A

Table 4. The top 25% of residues in 1ctsA at the interface with 1ctsA1. (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 5. res type disruptive mutations 271 G (KER)(FQMWHD)(NYLPI)(SVA) 274 H (TEQM)(KVCAG)(SNLPDI)(R) 242 N (Y)(FWH)(TER)(SVA) 273 L (T)(Y)(ER)(CG) Fig. 6. Residues in 1ctsA, at the interface with 1ctsA1, colored by their rela- 138 M (Y)(H)(TR)(SCDG) tive importance. 1ctsA1 is shown in backbone representation (See Appendix 272 P (Y)(T)(H)(R) for the coloring scheme for the protein chain 1ctsA.) 246 H (E)(Q)(D)(K) 268 G (KR)(E)(QH)(FMW) 239 E (FWH)(R)(YVA)(CG) 3 NOTES ON USING TRACE RESULTS 241 G (R)(E)(K)(H) 3.1 Coverage 243 V (R)(K)(YE)(H) 136 H (E)(Q)(T)(M) Trace results are commonly expressed in terms of coverage: the resi- 253 S (KR)(QH)(FMW)(E) due is important if its “coverage” is small - that is if it belongs to 240 G (R)(K)(E)(H) some small top percentage of residues [100% is all of the residues 250 L (Y)(R)(H)(T) in a chain], according to trace. The ET results are presented in the 251 V (R)(K)(E)(Y) form of a table, usually limited to top 25% percent of residues (or 56 S (K)(R)(QM)(FW) to some nearby percentage), sorted by the strength of the presumed 132 P (Y)(R)(H)(T) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 276 L (Y)(T)(H)(R) pressure on the residue.) Starting from the top of that list, mutating a 421 R (TD)(SEVCAG)(LPI)(Y) couple of residues should affect the protein somehow, with the exact 422 P (Y)(R)(H)(TE) effects to be determined experimentally. 254 A (R)(K)(E)(Y) 255 L (Y)(T)(R)(H) 3.2 Known substitutions 264 A (KR)(E)(Y)(QH) One of the table columns is “substitutions” - other amino acid types 44 G (R)(E)(K)(FWH) seen at the same position in the alignment. These amino acid types 270 A (K)(E)(R)(Q) may be interchangeable at that position in the protein, so if one wants 260 L (R)(KY)(H)(E) to affect the protein by a point mutation, they should be avoided. For 256 S (R)(K)(H)(Q) example if the substitutions are “RVK” and the original protein has 424 S (R)(K)(H)(FW) an R at that position, it is advisable to try anything, but RVK. Conver- 58 L (R)(Y)(H)(TKE) sely, when looking for substitutions which will not affect the protein, 420 E (FWH)(Y)(R)(VA) one may try replacing, R with K, or (perhaps more surprisingly), with 47 G (R)(K)(EH)(FW) V. The percentage of times the substitution appears in the alignment 423 K (Y)(FW)(T)(D) 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 5. List of disruptive mutations for the top 25% of residues in 1ctsA, guide - due to rounding errors these percentages often do not add up that are at the interface with 1ctsA1. to 100%. 3.3 Surface Figure 6 shows residues in 1ctsA colored by their importance, at the To detect candidates for novel functional interfaces, first we look for interface with 1ctsA1. residues that are solvent accessible (according to DSSP program) by

5 2 at least 10A˚ , which is roughly the area needed for one water mole- cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, COVERAGE the neighboring residues which are not surface accessible might be

equally important in maintaining the interaction specificity - they V should not be automatically dropped from consideration when choo- 100% 50% 30% 5% sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 3.4 Number of contacts Another column worth noting is denoted “noc/bb”; it tells the num- ber of contacts heavy atoms of the residue in question make across V the interface, as well as how many of them are realized through the RELATIVE IMPORTANCE backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . Fig. 7. Coloring scheme used to color residues by their relative importance. 3.5 Annotation If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” 2. their type appears. Annotations carried over from PDB are the following: site • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller • gaps Mutation suggestions are completely heuristic and based on comple- percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 7. disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will 4.3 Credits choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4 APPENDIX of residues, the average and range of the sequence lengths, and the 4.1 File formats alignment length (e.g. including gap characters). Also shown are Files with extension “ranks sorted” are the actual trace results. The some percent identities. A percent pairwise alignment identity is defi- fields in the table in this file: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two • alignment# number of the position in the alignment sequences. The ”average percent identity”, ”most related pair”, and • residue# residue number in the PDB file ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • type amino acid type seq” is calculated by finding the maximum pairwise identity (best • rank rank of the position according to older version of ET relative) for all N sequences, then finding the minimum of these N • variability has two subfields: numbers (hence, the most outlying sequence). alistat is copyrighted 1. number of different amino acids appearing in in this column by HHMI/Washington University School of Medicine, 1992-2001, of the alignment and freely distributed under the GNU General Public License.

6 4.3.2 CE To map ligand binding sites from different The viewer is self-unpacking and self-installing. Input files to be used source structures, report maker uses the CE program: with ETV (extension .etvx) can be found in the attachment to the http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) main report. ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.5 Citing this work The method used to rank residues and make predictions in this report 4.3.3 DSSP In this work a residue is considered solvent accessi- can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ˚ 2 ble if the DSSP program finds it exposed to water by at least 10A , Evolution-Entropy Hybrid Methods for Ranking of Protein Residues which is roughly the area needed for one water molecule to come in by Importance” J. Mol. Bio. 336: 1265-82. For the original version the contact with the residue. DSSP is copyrighted by W. Kabsch, C. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version tionary Trace Method Defines Binding Surfaces Common to Protein by [email protected] November 18,2002, Families” J. Mol. Bio. 257: 342-358. http://www.cmbi.kun.nl/gv/dssp/descrip.html. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- of service for comparative analysis of .” Bioinformatics gnment as a starting point for the analysis (sequences shorter than 22:1656-7. 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- 4.6 About report maker sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://swift.cmbi.kun.nl/swift/hssp/ king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.5 LaTex The text for this report was processed using LATEX; Houston. Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.7 Attachments The following files should accompany this report: 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), • 1ctsA.complex.pdb - coordinates of 1ctsA with all of its interac- ”MUSCLE: multiple sequence alignment with high accuracy and ting partners high throughput.” Nucleic Acids Research 32(5), 1792-97. • 1ctsA.etvx - ET viewer input file for 1ctsA http://www.drive5.com/muscle/ • 1ctsA.cluster report.summary - Cluster report summary for 1ctsA 4.3.7 Pymol The figures in this report were produced using • Pymol. The scripts can be found in the attachment. Pymol 1ctsA.ranks - Ranks file in sequence order for 1ctsA is an open-source application copyrighted by DeLano Scien- • 1ctsA.clusters - Cluster descriptions for 1ctsA tific LLC (2005). For more information about Pymol see • 1ctsA.msf - the multiple sequence alignment used for the chain http://pymol.sourceforge.net/. (Note for Windows 1ctsA users: the attached package needs to be unzipped for Pymol to read • 1ctsA.descr - description of sequences used in 1ctsA msf the scripts and launch the viewer.) • 1ctsA.ranks sorted - full listing of residues and their ranking for 4.4 Note about ET Viewer 1ctsA Dan Morgan from the Lichtarge lab has developed a visualization • 1ctsA.1ctsCIT1.if.pml - Pymol script for Figure 5 tool specifically for viewing trace results. If you are interested, please • 1ctsA.cbcvg - used by other 1ctsA – related pymol scripts visit: • 1ctsA.1ctsA1.if.pml - Pymol script for Figure 6 http://mammoth.bcm.tmc.edu/traceview/

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