Pages 1–6 1gnz Evolutionary trace report by report maker May 9, 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 1gnz): Title: Lectin i-b4 from griffonia simplicifolia (gs i-b4)metal free form CONTENTS Compound: Mol id: 1; molecule: lectin; chain: a; other details: lectin i-b4, metal free form 1 Introduction 1 Organism, scientific name: Griffonia Simplicifolia; 1gnz contains a single unique chain 1gnzA (236 residues long). 2 Chain 1gnzA 1 2.1 Q8W1R6 overview 1 2.2 Multiple sequence alignment for 1gnzA 1 2.3 Residue ranking in 1gnzA 1 2.4 Top ranking residues in 1gnzA and their position on 2 CHAIN 1GNZA the structure 2 2.1 Q8W1R6 overview 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Overlap with known functional surfaces at From SwissProt, id Q8W1R6, 100% identical to 1gnzA: 25% coverage. 2 Description: GSI-B4 isolectin (Fragment). 2.4.3 Possible novel functional surfaces at 25% Organism, scientific name: Griffonia simplicifolia. coverage. 3 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3 Notes on using trace results 4 eudicotyledons; rosids; eurosids I; Fabales; Fabaceae; Caesalpi- 3.1 Coverage 4 nioideae; Cercideae; Griffonia. 3.2 Known substitutions 4 3.3 Surface 4 3.4 Number of contacts 5 3.5 Annotation 5 2.2 Multiple sequence alignment for 1gnzA 3.6 Mutation suggestions 5 For the chain 1gnzA, the alignment 1gnzA.msf (attached) with 358 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 5 database, and fragments shorter than 75% of the query as well as 4.1 File formats 5 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 5 to this report, under the name of 1gnzA.msf. Its statistics, from the 4.3 Credits 5 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 4-121 in 1gnzA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 2. Residues 122-239 in 1gnzA colored by their relative importance. (See Appendix, Fig.8, for the coloring scheme.)

Fig. 3. Residues in 1gnzA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 358 Total number of residues: 79496 Smallest: 177 belong to. The clusters in Fig.4 are composed of the residues listed Largest: 236 Average length: 222.1 Alignment length: 236 Average identity: 34% Most related pair: 99% Most unrelated pair: 12% Most distant seq: 33%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 59% eukaryotic ( 59% plantae) sequences. (Descriptions of some sequences were not readily availa- ble.) The file containing the sequence descriptions can be found in the attachment, under the name 1gnzA.descr. 2.3 Residue ranking in 1gnzA The 1gnzA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1gnzA can be found in the file called 1gnzA.ranks sorted in the attachment. 2.4 Top ranking residues in 1gnzA and their position on the structure In the following we consider residues ranking among top 25% of Fig. 4. Residues in 1gnzA, colored according to the cluster they belong to: residues in the protein . Figure 3 shows residues in 1gnzA colored red, followed by blue and yellow are the largest clusters (see Appendix for by their importance: bright red and yellow indicate more conser- the coloring scheme). Clockwise: front, back, top and bottom views. The ved/important residues (see Appendix for the coloring scheme). A corresponding Pymol script is attached. 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. cluster size member color residues red 53 7,50,51,52,54,60,69,70,71,73 75,76,77,79,87,88,89,90,91 92,93,94,108,109,110,111,125 126,127,128,129,130,131,144 145,146,147,148,150,154,173 175,177,184,186,204,215,216 217,220,221,233,235

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

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. Phosphate ion binding site. Table 2 lists the top 25% of residues at the interface with 1gnzAPO41240 (phosphate ion). The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. Fig. 5. Residues in 1gnzA, at the interface with phosphate ion, colored by their relative importance. The ligand (phosphate ion) is colored green. Atoms res type subst’s cvg noc/ dist further than 30A˚ away from the geometric center of the ligand, as well as on ˚ (%) bb (A) the line of sight to the ligand were removed. (See Appendix for the coloring 96 P G(5) 0.18 11/3 3.75 scheme for the protein chain 1gnzA.) P(73)R V(1) S(9) A(4).K N(1) H(1)T

Table 2. The top 25% of residues in 1gnzA at the interface with phos- phate ion.(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. ) Fig. 6. A possible active surface on the chain 1gnzA. The larger cluster it belongs to is shown in blue. Table 3. res type disruptive 4, while Table 5 suggests possible disruptive replacements for these mutations residues (see Section 3.6). 96 P (Y)(R)(H)(E) Table 4. Table 3. List of disruptive mutations for the top 25% of residues in res type substitutions(%) cvg 1gnzA, that are at the interface with phosphate ion. 71 F F(95)W(2).STL 0.01 92 F F(97)L(1)TWS. 0.02 177 Y Y(88)F(10)A.ER 0.02 Figure 5 shows residues in 1gnzA colored by their importance, at the 127 V V(93)I(4)NL(1)C 0.05 interface with 1gnzAPO41240. A. 154 S KS(87)P(6)D(1)T 0.05 2.4.3 Possible novel functional surfaces at 25% coverage. One GVRFA group of residues is conserved on the 1gnzA surface, away from (or 144 H R(4)H(86)S(3) 0.06 susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1gnz. It is shown in Fig. 6. The right panel continued in next column shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table

3 Table 4. continued Table 5. continued res type substitutions(%) cvg res type disruptive .(1)LED(1)PQ(1) mutations G 51 R (D)(T)(E)(Y) 126 A A(88)YG(5)T(3)L 0.07 145 I (Y)(R)(H)(T) FHV.S 7 F (K)(E)(R)(TQ) 70 S S(77)D(9)N(8)T. 0.09 110 L (R)(Y)(T)(K) PAGH 184 L (R)(Y)(H)(K) 57 P P(85)Q(5)R(1) 0.10 54 Y (K)(Q)(R)(E) K(1).A(2)TLISG 96 P (Y)(R)(H)(E) 94 L L(63)I(24)F(1) 0.13 186 V (Y)(E)(R)(K) M(3)V(6).G 69 A (E)(KR)(Y)(D) 235 F F(91)L(3).(2)YP 0.13 58 V (Y)(R)(E)(K) SNVMR 51 R R(74)H(12)W(1)I 0.14 Table 5. Disruptive mutations for the surface patch in 1gnzA. S(1)GLVQ(4)T(1) .FKA Another group of surface residues is shown in Fig.7. The right panel 145 I I(58)V(33)TA 0.14 shows (in blue) the rest of the larger cluster this surface belongs to. L(2)M(1).DFNR 7 F F(84).(6)L(1) 0.16 D(2)VGIEAQY(1)W 110 L I(10)L(70)V(11) 0.17 W.(3)YM(1)PFHS 184 L L(78)M(1)V(5) 0.17 I(7)TF(2)QA(1). ESGY 54 Y Y(79)F(7)H(8)AL 0.18 SIP.CWE 96 P G(5)P(73)RV(1) 0.18 S(9)A(4).KN(1) H(1)T 186 V V(66)A(25)KT 0.21 I(1)L(1)M(2)R.F Fig. 7. Another possible active surface on the chain 1gnzA. The larger cluster 69 A A(67)S(8)V(3) 0.24 it belongs to is shown in blue. T(8)PL(2)YHF(1) .(1)R(2)IQ(1)CG The residues belonging to this surface ”patch” are listed in Table 58 V F(9)I(43)V(33) 0.25 6, while Table 7 suggests possible disruptive replacements for these L(10).(1)MQC residues (see Section 3.6).

Table 4. Residues forming surface ”patch” in 1gnzA. Table 6. res type substitutions(%) cvg 87 A G(41)A(45)XS(3) 0.12 Table 5. P(3)EV(1)T(2)DN res type disruptive .CQ mutations 88 D D(56)Y(9)E(6) 0.20 71 F (K)(E)(Q)(DR) G(3)H(12)V(2) 92 F (K)(E)(Q)(DR) S(1)A(1)N(1)T 177 Y (K)(Q)(M)(E) F(2)IQ. 127 V (Y)(R)(E)(K) 220 T T(67)S(22)L.(1) 0.21 154 S (R)(K)(H)(FQW) V(1)MG(1)N(2)RE 144 H (E)(T)(MD)(Q) AKI 126 A (K)(R)(E)(Q) 221 G G(81)E(1)A(3) 0.23 70 S (R)(K)(H)(Q) T(2).(1)NS(3) 57 P (Y)(R)(H)(T) Q(1)V(1)LRIMKY 94 L (R)(Y)(H)(T) 218 A A(74)PS(9)G(11) 0.25 235 F (E)(K)(T)(D) T(1).(1)NCLF continued in next column continued in next column

4 Table 6. continued should not be automatically dropped from consideration when choo- res type substitutions(%) cvg sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) Table 6. Residues forming surface ”patch” in 1gnzA. 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 Table 7. the interface, as well as how many of them are realized through the res type disruptive backbone atoms (if all or most contacts are through the backbone, mutations mutation presumably won’t have strong impact). Two heavy atoms 87 A (R)(Y)(K)(H) are considered to be “in contact” if their centers are closer than 5A˚ . 88 D (R)(H)(FW)(K) 220 T (R)(H)(K)(FW) 3.5 Annotation 221 G (R)(E)(K)(H) If the residue annotation is available (either from the pdb file or 218 A (R)(K)(E)(Y) from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 7. Disruptive mutations for the surface patch in 1gnzA. (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 GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino couple of residues should affect the protein somehow, with the exact acid, and they are grouped in round brackets if they appear equally effects to be determined experimentally. disruptive. From left to right, each bracketed group of amino acid types resembles more strongly the original (i.e. is, presumably, less 3.2 Known substitutions disruptive) These suggestions are tentative - they might prove disrup- One of the table columns is “substitutions” - other amino acid types tive to the fold rather than to the interaction. Many researcher will seen at the same position in the alignment. These amino acid types choose, however, the straightforward alanine mutations, especially in may be interchangeable at that position in the protein, so if one wants the beginning stages of their investigation. 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, one may try replacing, R with K, or (perhaps more surprisingly), with Files with extension “ranks sorted” are the actual trace results. The V. The percentage of times the substitution appears in the alignment fields in the table in this file: 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 to 100%. • type amino acid type • 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. 8. 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. 8. 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 • 1gnzA.msf - the multiple sequence alignment used for the chain The following files should accompany this report: 1gnzA • 1gnzA.descr - description of sequences used in 1gnzA msf • 1gnzA.complex.pdb - coordinates of 1gnzA with all of its • interacting partners 1gnzA.ranks sorted - full listing of residues and their ranking for 1gnzA • 1gnzA.etvx - ET viewer input file for 1gnzA • 1gnzA.1gnzAPO41240.if.pml - Pymol script for Figure 5 • 1gnzA.cluster report.summary - Cluster report summary for • 1gnzA 1gnzA.cbcvg - used by other 1gnzA – related pymol scripts • 1gnzA.ranks - Ranks file in sequence order for 1gnzA • 1gnzA.clusters - Cluster descriptions for 1gnzA

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