Pages 1–5 3gk5 Evolutionary trace report by report maker July 24, 2010

4.3.3 DSSP 4 4.3.4 HSSP 4 4.3.5 LaTex 5 4.3.6 Muscle 5 4.3.7 Pymol 5 4.4 Note about ET Viewer 5 4.5 Citing this work 5 4.6 About report maker 5 4.7 Attachments 5

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3gk5): Title: Crystal structure of rhodanese-related protein (tvg0868615) from volcanium, northeast structural genomics con- sortium target tvr109a Compound: Mol id: 1; molecule: uncharacterized rhodanese-related CONTENTS protein tvg0868615; chain: a; fragment: unp residues 101-200; engineered: yes 1 Introduction 1 Organism, scientific name: Gss1; 2 Chain 3gk5A 1 3gk5 contains a single unique chain 3gk5A (100 residues long). 2.1 Q97AG6 overview 1 2.2 Multiple sequence alignment for 3gk5A 1 2.3 Residue ranking in 3gk5A 1 2.4 Top ranking residues in 3gk5A and their position on the structure 1 2.4.1 Clustering of residues at 25% coverage. 1 2 CHAIN 3GK5A 2.4.2 Possible novel functional surfaces at 25% 2.1 Q97AG6 overview coverage. 2 From SwissProt, id Q97AG6, 100% identical to 3gk5A: Description: Hypothetical protein TVG0868615. 3 Notes on using trace results 3 Organism, scientific name: Thermoplasma volcanium. 3.1 Coverage 3 : ; ; ; Thermoplas- 3.2 Known substitutions 3 matales; ; Thermoplasma. 3.3 Surface 3 3.4 Number of contacts 3 3.5 Annotation 4 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 3gk5A 4 Appendix 4 For the chain 3gk5A, the alignment 3gk5A.msf (attached) with 876 4.1 File formats 4 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 4 database, and fragments shorter than 75% of the query as well as 4.3 Credits 4 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 4 to this report, under the name of 3gk5A.msf. Its statistics, from the 4.3.2 CE 4 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 101-200 in 3gk5A colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.)

Format: MSF Number of sequences: 876 Total number of residues: 79725 Smallest: 75 Largest: 100 Average length: 91.0 Alignment length: 100 Average identity: 31% Most related pair: 99% Most unrelated pair: 8% Most distant seq: 33%

Furthermore, <1% of residues show as conserved in this ali- Fig. 2. Residues in 3gk5A, colored by their relative importance. Clockwise: gnment. front, back, top and bottom views. The alignment consists of <1% eukaryotic ( <1% fungi), 12% prokaryotic, and 1% archaean sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 3gk5A.descr.

2.3 Residue ranking in 3gk5A The 3gk5A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 3gk5A can be found in the file called 3gk5A.ranks sorted in the attachment.

2.4 Top ranking residues in 3gk5A and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 3gk5A colored by their importance: bright red and yellow indicate more conser- ved/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 Fig. 3. Residues in 3gk5A, colored according to the cluster they belong to: in Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for the coloring scheme). Clockwise: front, back, top and bottom views. The Table 1. corresponding Pymol script is attached. cluster size member color residues red 25 120,121,122,123,126,130,135 2.4.2 Possible novel functional surfaces at 25% coverage. One 137,139,140,143,160,161,162 group of residues is conserved on the 3gk5A surface, away from (or 163,165,166,169,173,177,182 susbtantially larger than) other functional sites and interfaces reco- 183,185,186,190 gnizable in PDB entry 3gk5. It is shown in Fig. 4. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Table 1. Clusters of top ranking residues in 3gk5A. The residues belonging to this surface ”patch” are listed in Table

2 Table 2. continued res type substitutions(%) cvg 143 L V(5)I(21)F(7) 0.22 L(59)M(1)NAHDYE GPSR 162 H S(44)L(3)T(10) 0.23 M(3)A(5)K(1) V(10)ER(2)H(9) G(4)QINDYPF 137 N N(44)L(18)S(7) 0.24 H(18)T(1)RICF P(1)KA(1)YWQ(1) Fig. 4. A possible active surface on the chain 3gk5A. The larger cluster it .EG belongs to is shown in blue. 161 A R(36)K(10)G(3) 0.25 H(10)A(16)Q(14) E(1)D(1)N(1)S 2, while Table 3 suggests possible disruptive replacements for these L(1)MTY(1)VPWI residues (see Section 3.6).

Table 2. Table 2. Residues forming surface ”patch” in 3gk5A. res type substitutions(%) cvg 122 R R(98)CHSTL 0.02 163 G G(95)EMC(1)A(1) 0.04 PRSDY Table 3. 165 R R(90)H(1)S(1) 0.06 res type disruptive D(2)GVQTECNLIWM mutations KY 122 R (D)(E)(TY)(LPI) 126 E E(92)D(1)H(1) 0.07 163 G (R)(K)(E)(H) Q(1)GASLKVW 165 R (T)(D)(Y)(E) 185 G G(87)P(4)THEKNY 0.12 126 E (H)(FYW)(R)(CG) D(2)SQ.LRA 185 G (R)(KE)(FWH)(M) 139 P P(86)S(2)EYA(1) 0.13 139 P (Y)(R)(H)(T) LD(2)N(1)GMWTRV 169 A (R)(K)(E)(Y) KFIQH 135 S (R)(K)(H)(FW) 169 A A(80)V(11)T(1) 0.15 182 D (R)(H)(FW)(Y) M(1)IG(2)FS(1)H 130 G (R)(KE)(H)(D) PWL 123 E (H)(FYW)(R)(CG) 135 S A(61)S(25)IY 0.16 140 I (Y)(R)(T)(H) T(1)F(4)PEV(1)K 143 L (R)(Y)(H)(T) D(1)GMQHLNC 162 H (E)(T)(D)(Q) 182 D N(62)Y(2)R(1) 0.17 137 N (Y)(H)(T)(FW) D(7)S(9)H(3) 161 A (Y)(R)(K)(E) A(1)IL(1)T(1) E(1)MV(1)PKGCW Table 3. Disruptive mutations for the surface patch in 3gk5A. Q(1)F. 130 G G(63)A(14)V(2) 0.18 S(2)N(2)R(1) F(1)MC(4)LE(1) 3 NOTES ON USING TRACE RESULTS K(1)DTIQYWHP 123 E T(21)N(9)E(50)M 0.20 3.1 Coverage D(3)P(2)GQ(2)F Trace results are commonly expressed in terms of coverage: the resi- S(4)A(1)VK(1)LR due is important if its “coverage” is small - that is if it belongs to 140 I V(10)L(56)M(12) 0.21 some small top percentage of residues [100% is all of the residues Y(2)R(4)QSI(4) in a chain], according to trace. The ET results are presented in the F(2)HA(1)KGWTNP form of a table, usually limited to top 25% percent of residues (or continued in next column to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally.

3 3.2 Known substitutions One of the table columns is “substitutions” - other amino 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 COVERAGE example if the substitutions are “RVK” and the original protein has an R at that position, it is advisable to try anything, but RVK. Conver- V sely, when looking for substitutions which will not affect the protein, 100% 50% 30% 5% one may try replacing, R with K, or (perhaps more surprisingly), with 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 V to 100%. RELATIVE IMPORTANCE 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by Fig. 5. Coloring scheme used to color residues by their relative importance. 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 disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with 4.1 File formats the surface residues.) Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • alignment# number of the position in the alignment ber of contacts heavy atoms of the residue in question make across the interface, as well as how many of them are realized through the • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact). Two heavy atoms • rank rank of the position according to older version of ET are considered to be “in contact” if their centers are closer than 5A˚ . • variability has two subfields: 3.5 Annotation 1. number of different amino appearing in in this column If the residue annotation is available (either from the pdb file or of the alignment 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 Mutation suggestions are completely heuristic and based on comple- • gaps 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. 5.

4 4.3 Credits is an open-source application copyrighted by DeLano Scien- 4.3.1 Alistat alistat reads a multiple sequence alignment from the tific LLC (2005). For more information about Pymol see file and shows a number of simple statistics about it. These stati- http://pymol.sourceforge.net/. (Note for Windows stics include the format, the number of sequences, the total number users: the attached package needs to be unzipped for Pymol to read of residues, the average and range of the sequence lengths, and the the scripts and launch the viewer.) alignment length (e.g. including gap characters). Also shown are 4.4 Note about ET Viewer some percent identities. A percent pairwise alignment identity is defi- Dan Morgan from the Lichtarge lab has developed a visualization ned as (idents / MIN(len1, len2)) where idents is the number of tool specifically for viewing trace results. If you are interested, please exact identities and len1, len2 are the unaligned lengths of the two visit: sequences. The ”average percent identity”, ”most related pair”, and http://mammoth.bcm.tmc.edu/traceview/ ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant The viewer is self-unpacking and self-installing. Input files to be used seq” is calculated by finding the maximum pairwise identity (best with ETV (extension .etvx) can be found in the attachment to the relative) for all N sequences, then finding the minimum of these N main report. numbers (hence, the most outlying sequence). alistat is copyrighted by HHMI/Washington University School of Medicine, 1992-2001, 4.5 Citing this work and freely distributed under the GNU General Public License. The method used to rank residues and make predictions in this report can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of 4.3.2 CE To map ligand binding sites from different Evolution-Entropy Hybrid Methods for Ranking of Protein Residues source structures, report maker uses the CE program: by Importance” J. Mol. Bio. 336: 1265-82. For the original version http://cl.sdsc.edu/ . Shindyalov IN, Bourne PE (1998) of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- ”Protein structure alignment by incremental combinatorial extension tionary Trace Method Defines Binding Surfaces Common to Protein (CE) of the optimal path . Protein Engineering 11(9) 739-747. Families” J. Mol. Bio. 257: 342-358. 4.3.3 DSSP In this work a residue is considered solvent accessi- report maker itself is described in Mihalek I., I. Res and O. ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type which is roughly the area needed for one water molecule to come in of service for comparative analysis of proteins.” Bioinformatics the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 22:1656-7. Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 4.6 About report maker by [email protected] November 18,2002, report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://www.cmbi.kun.nl/gv/dssp/descrip.html. king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.4 HSSP Whenever available, report maker uses HSSP ali- Houston. gnment as a starting point for the analysis (sequences shorter than 75% of the query are taken out, however); R. Schneider, A. de 4.7 Attachments Daruvar, and C. Sander. ”The HSSP database of protein structure- The following files should accompany this report: sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. • 3gk5A.complex.pdb - coordinates of 3gk5A with all of its http://swift.cmbi.kun.nl/swift/hssp/ interacting partners • 3gk5A.etvx - ET viewer input file for 3gk5A 4.3.5 LaTex The text for this report was processed using LATEX; Leslie Lamport, “LaTeX: A Document Preparation System Addison- • 3gk5A.cluster report.summary - Cluster report summary for Wesley,” Reading, Mass. (1986). 3gk5A • 3gk5A.ranks - Ranks file in sequence order for 3gk5A 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), • 3gk5A.clusters - Cluster descriptions for 3gk5A ”MUSCLE: multiple sequence alignment with high accuracy and • 3gk5A.msf - the multiple sequence alignment used for the chain high throughput.” Nucleic Acids Research 32(5), 1792-97. 3gk5A • http://www.drive5.com/muscle/ 3gk5A.descr - description of sequences used in 3gk5A msf • 3gk5A.ranks sorted - full listing of residues and their ranking 4.3.7 Pymol The figures in this report were produced using for 3gk5A Pymol. The scripts can be found in the attachment. Pymol

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