Pages 1–7 3my6 Evolutionary trace report by report maker August 10, 2010

4.3.1 Alistat 5 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 3my6): Title: Crystal structure of the complex of type 1 ribosome inactiva protein with 7-methylguanine at 2.65 a resolution Compound: Mol id: 1; molecule: ribosome-inactivating pro- tein momordin i; chain: a; synonym: rrna n-glycosidase, alpha- momorcharin, alpha-mmc; ec: 3.2.2.22 Organism, scientific name: Balsamina; 3my6 contains a single unique chain 3my6A (246 residues long). CONTENTS 2 CHAIN 3MY6A 1 Introduction 1 2.1 P16094 overview 2 Chain 3my6A 1 From SwissProt, id P16094, 93% identical to 3my6A: 2.1 P16094 overview 1 Description: Ribosome-inactivating protein momordin I precursor 2.2 Multiple sequence alignment for 3my6A 1 (EC 3.2.2.22) (rRNA N-glycosidase) (Alpha-momorcharin) (Alpha- 2.3 Residue ranking in 3my6A 1 MMC). 2.4 Top ranking residues in 3my6A and their position on Organism, scientific name: (Bitter gourd) the structure 2 (Balsam pear). 2.4.1 Clustering of residues at 25% coverage. 2 : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.4.2 Overlap with known functional surfaces at Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 25% coverage. 2 eudicotyledons; ; eurosids I; ; ; 2.4.3 Possible novel functional surfaces at 25% Momordica. coverage. 3 Catalytic activity: Endohydrolysis of the N-glycosidic bond at one specific adenosine on the 28S rRNA. 3 Notes on using trace results 5 Similarity: Belongs to the ribosome-inactivating protein family. 3.1 Coverage 5 Type 1 RIP subfamily. 3.2 Known substitutions 5 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 5 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 5 EMBL outstation - the European Bioinformatics Institute. There are 3.5 Annotation 5 no restrictions on its use as long as its content is in no way modified 3.6 Mutation suggestions 5 and this statement is not removed.

4 Appendix 5 2.2 Multiple sequence alignment for 3my6A 4.1 File formats 5 For the chain 3my6A, the alignment 3my6A.msf (attached) with 117 4.2 Color schemes used 5 sequences was used. The alignment was downloaded from the HSSP 4.3 Credits 5 database, and fragments shorter than 75% of the query as well as

1 Lichtarge lab 2006 Pymol script for producing this figure can be found in the attachment.

Fig. 1. Residues 1-123 in 3my6A colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 2. Residues 124-246 in 3my6A colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.) duplicate sequences were removed. It can be found in the attachment to this report, under the name of 3my6A.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 3my6A, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 117 Total number of residues: 27610 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Smallest: 206 top 25% of all residues, this time colored according to clusters they Largest: 246 belong to. The clusters in Fig.4 are composed of the residues listed Average length: 236.0 in Table 1. Alignment length: 246 Average identity: 36% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 15% color residues Most distant seq: 30% red 56 14,17,21,22,25,34,35,36,37 49,61,63,65,66,68,69,70,71 72,73,74,81,82,105,107,109 Furthermore, <1% of residues show as conserved in this ali- 111,114,115,122,125,127,128 gnment. 132,139,148,153,155,156,157 The alignment consists of 55% eukaryotic ( 55% plantae) 160,161,162,163,164,166,167 sequences. (Descriptions of some sequences were not readily availa- 181,188,189,190,192,195,196 ble.) The file containing the sequence descriptions can be found in 199,200 the attachment, under the name 3my6A.descr. blue 4 207,209,215,225 2.3 Residue ranking in 3my6A Table 1. Clusters of top ranking residues in 3my6A. The 3my6A sequence is shown in Figs. 1–2, with each residue colo- red according to its estimated importance. The full listing of residues in 3my6A can be found in the file called 3my6A.ranks sorted in the 2.4.2 Overlap with known functional surfaces at 25% coverage. attachment. The name of the ligand is composed of the source PDB identifier 2.4 Top ranking residues in 3my6A and their position on and the heteroatom name used in that file. MY6 binding site. Table 2 lists the top 25% of residues at the the structure interface with 3my6AMY6247 (my6). The following table (Table In the following we consider residues ranking among top 25% of 3) suggests possible disruptive replacements for these residues (see residues in the protein . Figure 3 shows residues in 3my6A colored Section 3.6). by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A

2 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 111 Y (K)(Q)(EM)(NR) 163 R (T)(D)(SYEVCAG)(LPI) 160 E (H)(YR)(FW)(CG) 70 Y (K)(EQMR)(NLPDI)(VA) 155 I (YR)(H)(T)(KE) 69 V (KER)(Y)(QD)(H) 109 G (R)(K)(FWH)(E) 71 I (R)(Y)(T)(KEH) 72 M (Y)(H)(R)(T)

Table 3. List of disruptive mutations for the top 25% of residues in 3my6A, that are at the interface with MY6.

Fig. 4. Residues in 3my6A, colored according to the cluster they belong to: 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 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 111 Y Y(99)F 0.01 14/2 3.75 163 R R(98)KH 0.02 5/0 3.58 site 160 E E(98)AL 0.03 3/0 3.71 70 Y Y(94)G 0.07 76/13 3.39 site N(4)A 155 I I(95) 0.07 15/0 3.06 site V(2)L. 69 V V(24) 0.20 2/2 4.21 L(39) A(35)F 109 G S(19) 0.20 27/27 3.17 G(74) N(1) Fig. 5. Residues in 3my6A, at the interface with MY6, colored by their rela- E(3)T tive importance. The ligand (MY6) is colored green. Atoms further than 30A˚ 71 I L(10) 0.24 26/20 3.11 site away from the geometric center of the ligand, as well as on the line of sight V(72) to the ligand were removed. (See Appendix for the coloring scheme for the I(16)F protein chain 3my6A.) 72 M L(4) 0.24 1/0 4.34 V(61) Figure 5 shows residues in 3my6A colored by their importance, at M(26) the interface with 3my6AMY6247. I(3) A(3)E 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 3my6A surface, away from (or Table 2. The top 25% of residues in 3my6A at the interface with susbtantially larger than) other functional sites and interfaces reco- MY6.(Field names: res: residue number in the PDB entry; type: amino acid gnizable in PDB entry 3my6. It is shown in Fig. 6. The right panel type; substs: substitutions seen in the alignment; with the percentage of each shows (in blue) the rest of the larger cluster this surface belongs to.

3 Table 4. continued res type substitutions(%) cvg antn 109 G S(19)G(74)N(1) 0.20 E(3)T 49 L L(78)F(1)A(1)I 0.21 T(5)V(5)S(1) R(1)KY 166 Y Y(70)P(7)A(4) 0.21 F(13)IHRVC 207 G G(83)QD(9)K(2)E 0.21 S(2) 115 Q E(68)Q(23)F(1) 0.22 Fig. 6. A possible active surface on the chain 3my6A. The larger cluster it L(1)AYRG(1) belongs to is shown in blue. 225 I V(66)L(8)I(18) 0.22 W(1).(3)A 82 F Y(5)F(73)H(5) 0.23 The residues belonging to this surface ”patch” are listed in Table L(4)I(9)T 4, while Table 5 suggests possible disruptive replacements for these 189 E Q(7)E(90)IK 0.23 residues (see Section 3.6). 71 I L(10)V(72)I(16) 0.24 site Table 4. F res type substitutions(%) cvg antn 139 L F(1)L(71)V(13) 0.24 111 Y Y(99)F 0.01 M(2)I(9)S 192 W W(100) 0.01 61 T T(75)I(1)E(5) 0.25 163 R R(98)KH 0.02 site S(12)Q(3)NA 160 E E(98)AL 0.03 190 N N(65)T(12)A 0.25 122 R R(97)IGK 0.04 E(15)RKLD(2) 127 I L(97)I(2) 0.04 215 L L(94)I(4)M 0.05 Table 4. Residues forming surface ”patch” in 3my6A. 181 P KP(94)V(1)AE(1) 0.06 N 188 L L(87)M(9)Y(2)F 0.06 Table 5. 70 Y Y(94)GN(4)A 0.07 site res type disruptive 155 I I(95)V(2)L. 0.07 site mutations 22 R R(95)Q(3)H 0.08 111 Y (K)(Q)(EM)(NR) 74 Y F(18)Y(80)C 0.09 192 W (KE)(TQD)(SNCRG)(M) 209 F F(81)L(12)I(5)Q 0.11 163 R (T)(D)(SYEVCAG)(LPI) 68 N N(88)D(5)T(1) 0.12 160 E (H)(YR)(FW)(CG) S(4) 122 R (TYD)(E)(S)(FVCAWG) 107 Y F(58)Y(37)CL 0.13 127 I (YR)(TH)(SKECG)(FQWD) V(1) 215 L (Y)(R)(TH)(CG) 25 L L(57)V(18)A(17) 0.15 181 P (Y)(R)(H)(T) F(4)I(1)K 188 L (R)(TY)(K)(EH) 81 Y Y(82)H(9)F(7)N 0.15 70 Y (K)(EQMR)(NLPDI)(VA) 65 D D(67)R(13)N(10) 0.16 155 I (YR)(H)(T)(KE) K(2)S(2)Q(1) 22 R (T)(D)(SYE)(VCAG) T(1) 74 Y (K)(Q)(M)(E) 66 V V(72)R(21)L(2) 0.16 209 F (TE)(K)(DR)(SCG) K(1)AG 68 N (FYWH)(R)(TEVMA)(KCG) 125 I T(5)I(64)V(23) 0.17 107 Y (K)(Q)(E)(R) F(2)A(1)L(1) 25 L (Y)(R)(T)(H) 153 V V(76)I(17)LG(3) 0.19 81 Y (K)(EQM)(VAR)(NLPDI) .A 65 D (FW)(HR)(Y)(VA) 47 Y F(52)Y(36)L(4) 0.20 66 V (Y)(E)(R)(D) R(2)NAVH 125 I (R)(Y)(K)(H) continued in next column 153 V (R)(Y)(KE)(H) continued in next column

4 Table 5. continued should not be automatically dropped from consideration when choo- res type disruptive sing the set for mutagenesis. (Especially if they form a cluster with mutations the surface residues.) 47 Y (K)(E)(Q)(M) 109 G (R)(K)(FWH)(E) 3.4 Number of contacts 49 L (R)(Y)(H)(E) Another column worth noting is denoted “noc/bb”; it tells the num- 166 Y (K)(E)(Q)(MR) ber of contacts heavy atoms of the residue in question make across 207 G (FW)(R)(H)(KY) the interface, as well as how many of them are realized through the 115 Q (Y)(TH)(FW)(SCG) backbone atoms (if all or most contacts are through the backbone, 225 I (R)(Y)(T)(H) mutation presumably won’t have strong impact). Two heavy atoms 82 F (K)(E)(Q)(R) are considered to be “in contact” if their centers are closer than 5A˚ . 189 E (FYWH)(CG)(TVA)(R) 71 I (R)(Y)(T)(KEH) 3.5 Annotation 139 L (R)(Y)(H)(T) If the residue annotation is available (either from the pdb file or 61 T (R)(H)(K)(FW) from other sources), another column, with the header “annotation” 190 N (Y)(FWH)(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 3my6A. 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 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 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- 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 example if the substitutions are “RVK” and the original protein has 4 APPENDIX an R at that position, it is advisable to try anything, but RVK. Conver- 4.1 File formats 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 is given in the immediately following bracket. No percentage is given • alignment# number of the position in the alignment in the cases when it is smaller than 1%. This is meant to be a rough • residue# residue number in the PDB file 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 3.3 Surface • variability has two subfields: To detect candidates for novel functional interfaces, first we look for 1. number of different amino acids appearing in in this column residues that are solvent accessible (according to DSSP program) by of the alignment 2 at least 10A˚ , which is roughly the area needed for one water mole- 2. their type cule to come in the contact with the residue. Furthermore, we require • that these residues form a “cluster” of residues which have neighbor rho ET score - the smaller this value, the lesser variability of within 5A˚ from any of their heavy atoms. this position across the branches of the tree (and, presumably, Note, however, that, if our picture of protein evolution is correct, the greater the importance for the protein) the neighboring residues which are not surface accessible might be • cvg coverage - percentage of the residues on the structure which equally important in maintaining the interaction specificity - they have this rho or smaller

5 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 by [email protected] November 18,2002, COVERAGE http://www.cmbi.kun.nl/gv/dssp/descrip.html.

V 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 100% 50% 30% 5% 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- sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. http://swift.cmbi.kun.nl/swift/hssp/ V 4.3.5 LaTex The text for this report was processed using LATEX; RELATIVE IMPORTANCE Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). Fig. 7. Coloring scheme used to color residues by their relative importance. 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), ”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 file and shows a number of simple statistics about it. These stati- http://mammoth.bcm.tmc.edu/traceview/ 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. by HHMI/Washington University School of Medicine, 1992-2001, report maker itself is described in Mihalek I., I. Res and O. and freely distributed under the GNU General Public License. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.2 CE To map ligand binding sites from different of service for comparative analysis of proteins.” Bioinformatics source structures, report maker uses the CE program: 22:1656-7. http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) 4.6 About report maker ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.3 DSSP In this work a residue is considered solvent accessi- is copyrighted by Lichtarge Lab, Baylor College of Medicine, ble if the DSSP program finds it exposed to water by at least 10A˚ 2, Houston.

6 4.7 Attachments • 3my6A.msf - the multiple sequence alignment used for the chain The following files should accompany this report: 3my6A • 3my6A.descr - description of sequences used in 3my6A msf • 3my6A.complex.pdb - coordinates of 3my6A with all of its • interacting partners 3my6A.ranks sorted - full listing of residues and their ranking for 3my6A • 3my6A.etvx - ET viewer input file for 3my6A • 3my6A.3my6AMY6247.if.pml - Pymol script for Figure 5 • 3my6A.cluster report.summary - Cluster report summary for • 3my6A 3my6A.cbcvg - used by other 3my6A – related pymol scripts • 3my6A.ranks - Ranks file in sequence order for 3my6A • 3my6A.clusters - Cluster descriptions for 3my6A

7