Pages 1–8 1vbf Evolutionary trace report by report maker July 2, 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 8 4.6 About report maker 8 4.7 Attachments 8

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1vbf): Title: Crystal structure of protein l-isoaspartate o- methyltransferase homologue from tokodaii CONTENTS Compound: Mol id: 1; molecule: 231aa long hypothetical protein-l- isoaspartate o- methyltransferase; chain: a, b, c, d; synonym: protein 1 Introduction 1 l-isoaspartate o-methyltransferase; ec: 2.1.1.77; engineered: yes Organism, scientific name: Sulfolobus Tokodaii; 2 Chain 1vbfB 1 1vbf contains a single unique chain 1vbfB (226 residues long) and 2.1 Q972K9 overview 1 its homologues 1vbfA, 1vbfD, and 1vbfC. 2.2 Multiple sequence alignment for 1vbfB 1 2.3 Residue ranking in 1vbfB 1 2.4 Top ranking residues in 1vbfB and their position on the structure 2 2.4.1 Clustering of residues at 25% coverage. 2 2 CHAIN 1VBFB 2.4.2 Overlap with known functional surfaces at 2.1 Q972K9 overview 25% coverage. 2 2.4.3 Possible novel functional surfaces at 25% From SwissProt, id Q972K9, 92% identical to 1vbfB: coverage. 5 Description: Hypothetical protein ST1123. Organism, scientific name: Sulfolobus tokodaii. 3 Notes on using trace results 6 : ; ; ; ; 3.1 Coverage 6 ; Sulfolobus. 3.2 Known substitutions 6 3.3 Surface 6 3.4 Number of contacts 6 3.5 Annotation 6 2.2 Multiple sequence alignment for 1vbfB 3.6 Mutation suggestions 6 For the chain 1vbfB, the alignment 1vbfB.msf (attached) with 327 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 7 database, and fragments shorter than 75% of the query as well as 4.1 File formats 7 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 7 to this report, under the name of 1vbfB.msf. Its statistics, from the 4.3 Credits 7 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-113 in 1vbfB colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

Fig. 2. Residues 114-226 in 1vbfB colored by their relative importance. (See Appendix, Fig.9, for the coloring scheme.)

Fig. 3. Residues in 1vbfB, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 327 Total number of residues: 61703 Smallest: 76 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 226 Average length: 188.7 Alignment length: 226 Average identity: 41% Most related pair: 99% Most unrelated pair: 10% Most distant seq: 30%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of <1% eukaryotic ( <1% vertebrata), 14% prokaryotic, and 6% 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 1vbfB.descr. 2.3 Residue ranking in 1vbfB The 1vbfB sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1vbfB can be found in the file called 1vbfB.ranks sorted in the attachment. 2.4 Top ranking residues in 1vbfB and their position on the structure Fig. 4. Residues in 1vbfB, colored according to the cluster they belong to: In the following we consider residues ranking among top 25% of resi- red, followed by blue and yellow are the largest clusters (see Appendix for dues in the protein . Figure 3 shows residues in 1vbfB colored by their the coloring scheme). Clockwise: front, back, top and bottom views. The importance: bright red and yellow indicate more conserved/important corresponding Pymol script is attached. residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1. 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the top 25% of all residues, this time colored according to clusters they

2 Table 1. Table 3. cluster size member res type disruptive color residues mutations red 55 24,25,28,37,38,45,46,47,51 46 P (R)(Y)(H)(T) 52,53,54,55,56,62,66,74,75 47 I (R)(Y)(K)(E) 76,78,79,80,81,82,83,84,85 40 H (E)(T)(Q)(D) 86,88,95,99,100,104,108,112 45 L (R)(Y)(H)(K) 124,125,128,134,135,136,138 141,142,148,151,154,155,159 Table 3. List of disruptive mutations for the top 25% of residues in 1vbfB, 164,166,171,191,193,194 that are at the interface with 1vbfD2.

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

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 1vbfD2.By analogy with 1vbfC – 1vbfD2 inter- face. Table 2 lists the top 25% of residues at the interface with 1vbfD2. 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˚ ) 46 P G(7) 0.17 38/7 2.98 P(79)DQ TNE(2)Y .(1) S(3) A(1)RV 47 I Y(4) 0.20 28/22 2.39 I(86)F L(3)G .(1)HMC S Fig. 5. Residues in 1vbfB, at the interface with 1vbfD2, colored by their rela- 40 H D(68) 0.22 4/0 4.20 tive importance. 1vbfD2 is shown in backbone representation (See Appendix N(22)G for the coloring scheme for the protein chain 1vbfB.) Y(1) H(2) L(1)SKP Figure 5 shows residues in 1vbfB colored by their importance, at the .(1)AI interface with 1vbfD2. 45 L I(8) 0.23 1/0 4.39 Interface with 1vbfA.Table 4 lists the top 25% of residues at the V(2) interface with 1vbfA. The following table (Table 5) suggests possible L(80) disruptive replacements for these residues (see Section 3.6). F(4) Table 4. .(2)PMT res type subst’s cvg noc/ dist AY (%) bb (A˚ ) 148 L P(92)DS 0.07 2/2 4.59 Table 2. The top 25% of residues in 1vbfB at the interface with 1vbfD2. IV(1) (Field names: res: residue number in the PDB entry; type: amino acid type; L(2).AH substs: substitutions seen in the alignment; with the percentage of each type M in the bracket; noc/bb: number of contacts with the ligand, with the number of 166 G G(88)S 0.16 8/8 3.10 contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) E(1) A(2)LWI KMQP continued in next column

3 Table 4. continued Table 6. res type subst’s cvg noc/ dist res type subst’s cvg noc/ dist (%) bb (A˚ ) (%) bb (A˚ ) .(1)TDV 95 V TV(88) 0.12 9/9 3.71 R L(6)AN I(4) Table 4. The top 25% of residues in 1vbfB at the interface with 1vbfA. (Field names: res: residue number in the PDB entry; type: amino acid type; Table 6. The top 25% of residues in 1vbfB at the interface with 1vbfC. substs: substitutions seen in the alignment; with the percentage of each type (Field names: res: residue number in the PDB entry; type: amino acid type; in the bracket; noc/bb: number of contacts with the ligand, with the number of substs: substitutions seen in the alignment; with the percentage of each type contacts realized through backbone atoms given in the bracket; dist: distance in the bracket; noc/bb: number of contacts with the ligand, with the number of of closest apporach to the ligand. ) contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 5. res type disruptive Table 7. mutations res type disruptive 148 L (R)(Y)(H)(T) mutations 166 G (R)(K)(E)(H) 95 V (R)(Y)(KE)(H)

Table 5. List of disruptive mutations for the top 25% of residues in 1vbfB, Table 7. List of disruptive mutations for the top 25% of residues in 1vbfB, that are at the interface with 1vbfA. that are at the interface with 1vbfC.

Fig. 6. Residues in 1vbfB, at the interface with 1vbfA, colored by their rela- Fig. 7. Residues in 1vbfB, at the interface with 1vbfC, colored by their rela- tive importance. 1vbfA is shown in backbone representation (See Appendix tive importance. 1vbfC is shown in backbone representation (See Appendix for the coloring scheme for the protein chain 1vbfB.) for the coloring scheme for the protein chain 1vbfB.)

Figure 6 shows residues in 1vbfB colored by their importance, at the Figure 7 shows residues in 1vbfB colored by their importance, at the interface with 1vbfA. interface with 1vbfC. Interface with 1vbfC.Table 6 lists the top 25% of residues at the interface with 1vbfC. The following table (Table 7) suggests possible 2.4.3 Possible novel functional surfaces at 25% coverage. One disruptive replacements for these residues (see Section 3.6). group of residues is conserved on the 1vbfB surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1vbf. It is shown in Fig. 8. The right panel shows (in blue) the rest of the larger cluster this surface belongs to.

4 Table 8. continued res type substitutions(%) cvg WIKMQP.(1)TDVR 46 P G(7)P(79)DQTN 0.17 E(2)Y.(1)S(3) A(1)RV 51 I I(7)Q(72)A(1) 0.18 S(1)V(1)Y(1) N(4).(4)RE(1)LF MKGT 52 N S(7)T(73)W(1) 0.18 N(1)Q(8)FV(1)D. Fig. 8. A possible active surface on the chain 1vbfB. The larger cluster it RLGIA belongs to is shown in blue. 191 V V(76).(3)C(7) 0.19 F(7)ILTA(1)RGSK 47 I Y(4)I(86)FL(3)G 0.20 The residues belonging to this surface ”patch” are listed in Table .(1)HMCS 8, while Table 9 suggests possible disruptive replacements for these 171 Q Q(85)T(1)I(2)GS 0.20 residues (see Section 3.6). HPR(1).(2)E(1)L Table 8. DMNA res type substitutions(%) cvg 100 I GR(73)H(3)I(14) 0.21 78 G G(100) 0.00 Y(1)V(2)FL(1)TQ 25 R R(97)QF.(2) 0.02 DC(1)EM 80 G G(98)A(1) 0.03 40 H D(68)N(22)GY(1) 0.22 125 G G(95)EL.QDAIK 0.03 H(2)L(1)SKP.(1) 28 F F(94)Y(2)TR.(2) 0.04 AI 124 D D(95)SN(2)P.(1) 0.04 194 G .(8)V(81)Q(1) 0.22 EG G(2)ATLMK(1)F 128 G G(94)VL(1)I.RTA 0.06 S(1)R EKF 24 D D(11)P(78)E(1) 0.23 37 A A(91)P(3)K(1) 0.07 R(1)HNS.(2)K(1) S(1)V.(1)N A(1) 99 E IE(93)D(5)S 0.07 45 L I(8)V(2)L(80) 0.23 108 A A(87)T(6)S(3)FP 0.09 F(4).(2)PMTAY VLGD 141 W G(5)A(9)T(70) 0.23 79 T S(7)T(81)A(7) 0.10 S(6)HW(3)E.(1)V C(1)GPL CPYNFQ 142 A A(91)C(4)GS(1)R 0.10 112 L L(77)F(17)YE 0.24 .VIT M(1)HI(1)SNV 54 T P(7)S(74)T(4) 0.11 53 T A(4)I(68)M(2) 0.25 I(7)L(2)E.AKHQ V(3)CT(11)L(3)S 81 I S(84)T(3)C(6) 0.11 Q(2)FK.G L(2)HI(2)Y.V 55 A H(5)Q(59)A(14) 0.25 38 Y Y(87)F(2)GHW(5) 0.12 S(11)G(1)Y(1)D SRTI.(1)QMN P(1)KVF.ILRTE 84 Y L(5)A(10)Q(64) 0.14 H(7)N(5)Y(1) Table 8. Residues forming surface ”patch” in 1vbfB. M(1)S(1)T(1)VGF I 193 F .(4)F(88)Y(4)RN 0.14 Table 9. LTSIM res type disruptive 104 X I(3)L(90)EV(1)A 0.16 mutations RFM(1)NSX 78 G (KER)(FQMWHD)(NYLPI)(SVA) 166 G G(88)SE(1)A(2)L 0.16 25 R (T)(D)(SCG)(YEVA) continued in next column 80 G (KER)(QHD)(FYMW)(N) 125 G (R)(H)(FW)(KY) continued in next column

5 Table 9. continued one may try replacing, R with K, or (perhaps more surprisingly), with res type disruptive V. The percentage of times the substitution appears in the alignment mutations is given in the immediately following bracket. No percentage is given 28 F (KE)(QD)(TN)(SCG) in the cases when it is smaller than 1%. This is meant to be a rough 124 D (R)(H)(FW)(Y) guide - due to rounding errors these percentages often do not add up 128 G (R)(E)(K)(H) to 100%. 37 A (Y)(R)(E)(H) 99 E (H)(FWR)(Y)(CG) 108 A (R)(K)(YE)(H) 3.3 Surface 79 T (R)(K)(H)(Q) To detect candidates for novel functional interfaces, first we look for 142 A (KR)(E)(Y)(H) residues that are solvent accessible (according to DSSP program) by 2 54 T (R)(K)(H)(FW) at least 10A˚ , which is roughly the area needed for one water mole- 81 I (R)(Y)(K)(H) cule to come in the contact with the residue. Furthermore, we require 38 Y (K)(Q)(EM)(R) that these residues form a “cluster” of residues which have neighbor 84 Y (K)(R)(Q)(E) within 5A˚ from any of their heavy atoms. 193 F (E)(K)(TD)(Q) Note, however, that, if our picture of protein evolution is correct, 104 X (Y)(R)(KE)(H) the neighboring residues which are not surface accessible might be 166 G (R)(K)(E)(H) equally important in maintaining the interaction specificity - they 46 P (R)(Y)(H)(T) should not be automatically dropped from consideration when choo- 51 I (Y)(R)(H)(T) sing the set for mutagenesis. (Especially if they form a cluster with 52 N (Y)(H)(TR)(FW) the surface residues.) 191 V (E)(Y)(R)(K) 47 I (R)(Y)(K)(E) 171 Q (Y)(H)(FW)(T) 3.4 Number of contacts 100 I (R)(Y)(H)(T) Another column worth noting is denoted “noc/bb”; it tells the num- 40 H (E)(T)(Q)(D) ber of contacts heavy atoms of the residue in question make across 194 G (E)(R)(K)(H) the interface, as well as how many of them are realized through the 24 D (R)(FW)(H)(Y) backbone atoms (if all or most contacts are through the backbone, 45 L (R)(Y)(H)(K) mutation presumably won’t have strong impact). Two heavy atoms 141 W (K)(E)(Q)(D) are considered to be “in contact” if their centers are closer than 5A˚ . 112 L (R)(Y)(T)(H) 53 T (R)(K)(H)(FW) 55 A (R)(Y)(K)(E) 3.5 Annotation If the residue annotation is available (either from the pdb file or Table 9. Disruptive mutations for the surface patch in 1vbfB. 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 bond forming residue), hb (hydrogen bond forming residue, jb (james 3 NOTES ON USING TRACE RESULTS bond forming residue), and sb (for salt bridge forming residue). 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to 3.6 Mutation suggestions some small top percentage of residues [100% is all of the residues Mutation suggestions are completely heuristic and based on comple- in a chain], according to trace. The ET results are presented in the mentarity with the substitutions found in the alignment. Note that form of a table, usually limited to top 25% percent of residues (or they are meant to be disruptive to the interaction of the protein to some nearby percentage), sorted by the strength of the presumed with its ligand. The attempt is made to complement the following evolutionary pressure. (I.e., the smaller the coverage, the stronger the properties: small [AV GSTC], medium [LPNQDEMIK], large pressure on the residue.) Starting from the top of that list, mutating a [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- couple of residues should affect the protein somehow, with the exact tively [KHR], or negatively [DE] charged, aromatic [WFYH], effects to be determined experimentally. long aliphatic chain [EKRQM], OH-group possession [SDETY ], and NH2 group possession [NQRK]. The suggestions are listed 3.2 Known substitutions according to how different they appear to be from the original amino One of the table columns is “substitutions” - other amino acid types acid, and they are grouped in round brackets if they appear equally seen at the same position in the alignment. These amino acid types disruptive. From left to right, each bracketed group of amino acid may be interchangeable at that position in the protein, so if one wants types resembles more strongly the original (i.e. is, presumably, less to affect the protein by a point mutation, they should be avoided. For disruptive) These suggestions are tentative - they might prove disrup- example if the substitutions are “RVK” and the original protein has tive to the fold rather than to the interaction. Many researcher will an R at that position, it is advisable to try anything, but RVK. Conver- choose, however, the straightforward alanine mutations, especially in sely, when looking for substitutions which will not affect the protein, the beginning stages of their investigation.

6 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 exact identities and len1, len2 are the unaligned lengths of the two COVERAGE sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum,

V and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant 100% 50% 30% 5% 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 by HHMI/Washington University School of Medicine, 1992-2001, and freely distributed under the GNU General Public License.

V 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: RELATIVE IMPORTANCE http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension Fig. 9. Coloring scheme used to color residues by their relative importance. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 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 • 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 The following color scheme is used in figures with residues colored http://www.drive5.com/muscle/ by cluster size: black is a single-residue cluster; clusters composed of more than one residue colored according to this hierarchy (ordered 4.3.7 Pymol The figures in this report were produced using by descending size): red, blue, yellow, green, purple, azure, tur- Pymol. The scripts can be found in the attachment. Pymol quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, is an open-source application copyrighted by DeLano Scien- bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, tific LLC (2005). For more information about Pymol see DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, http://pymol.sourceforge.net/. (Note for Windows tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. users: the attached package needs to be unzipped for Pymol to read The colors used to distinguish the residues by the estimated the scripts and launch the viewer.) evolutionary pressure they experience can be seen in Fig. 9. 4.4 Note about ET Viewer 4.3 Credits Dan Morgan from the Lichtarge lab has developed a visualization 4.3.1 Alistat alistat reads a multiple sequence alignment from the tool specifically for viewing trace results. If you are interested, please file and shows a number of simple statistics about it. These stati- visit: stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the http://mammoth.bcm.tmc.edu/traceview/

7 The viewer is self-unpacking and self-installing. Input files to be used 4.7 Attachments with ETV (extension .etvx) can be found in the attachment to the The following files should accompany this report: main report. • 1vbfB.complex.pdb - coordinates of 1vbfB with all of its inter- acting partners 4.5 Citing this work • 1vbfB.etvx - ET viewer input file for 1vbfB 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 1vbfB.cluster report.summary - Cluster report summary for Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 1vbfB by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1vbfB.ranks - Ranks file in sequence order for 1vbfB of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1vbfB.clusters - Cluster descriptions for 1vbfB tionary Trace Method Defines Binding Surfaces Common to Protein • 1vbfB.msf - the multiple sequence alignment used for the chain Families” J. Mol. Bio. 257: 342-358. 1vbfB report maker itself is described in Mihalek I., I. Res and O. • Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 1vbfB.descr - description of sequences used in 1vbfB msf of service for comparative analysis of proteins.” Bioinformatics • 1vbfB.ranks sorted - full listing of residues and their ranking for 22:1656-7. 1vbfB • 1vbfB.1vbfD2.if.pml - Pymol script for Figure 5 4.6 About report maker • 1vbfB.cbcvg - used by other 1vbfB – related pymol scripts report maker was written in 2006 by Ivana Mihalek. The 1D ran- • 1vbfB.1vbfA.if.pml - Pymol script for Figure 6 king visualization program was written by Ivica Res.ˇ report maker • 1vbfB.1vbfC.if.pml - Pymol script for Figure 7 is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston.

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