Pages 1–6 1iq9 Evolutionary trace report by report maker April 18, 2010

4.3.1 Alistat 5 4.3.2 CE 5 4.3.3 DSSP 5 4.3.4 HSSP 5 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 1iq9): Title: Crystal structure at 1.8 a of toxin a from nigricollis venom Compound: Mol id: 1; molecule: alpha-neurotoxin; chain: a; synonym: short neurotoxin 1 Organism, scientific name: Naja Nigricollis; 1iq9 contains a single unique chain 1iq9A (61 residues long).

2 CHAIN 1IQ9A CONTENTS 2.1 P01426 overview 1 Introduction 1 From SwissProt, id P01426, 100% identical to 1iq9A: Description: Short neurotoxin 1 (Neurotoxin alpha). 2 Chain 1iq9A 1 Organism, scientific name: Naja pallida (Red spitting cobra). 2.1 P01426 overview 1 : Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.2 Multiple sequence alignment for 1iq9A 1 Euteleostomi; Lepidosauria; ; Scleroglossa; Serpentes; 2.3 Residue ranking in 1iq9A 1 Colubroidea; ; Elapinae; Naja. 2.4 Top ranking residues in 1iq9A and their position on Function: Produces peripheral paralysis by blocking neuromuscular the structure 2 transmission at the postsynaptic site. Binds to the muscular nicotinic 2.4.1 Clustering of residues at 25% coverage. 2 acetylcholine receptor. 2.4.2 Overlap with known functional surfaces at Subcellular location: Secreted. 25% coverage. 2 Tissue specificity: Expressed by the venom gland. 2.4.3 Possible novel functional surfaces at 25% Toxic dose: LD(50) is 0.036 mg/kg by subcutaneous injection. coverage. 3 Similarity: Belongs to the toxin family. Type I alpha- neuro- toxin subfamily. 3 Notes on using trace results 4 Caution: The venom of this snake was originally thought to be that 3.1 Coverage 4 of N.nigricollis. 3.2 Known substitutions 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.3 Surface 4 collaboration between the Swiss Institute of Bioinformatics and the 3.4 Number of contacts 4 EMBL outstation - the European Bioinformatics Institute. There are 3.5 Annotation 4 no restrictions on its use as long as its content is in no way modified 3.6 Mutation suggestions 4 and this statement is not removed.

4 Appendix 4 2.2 Multiple sequence alignment for 1iq9A 4.1 File formats 4 For the chain 1iq9A, the alignment 1iq9A.msf (attached) with 291 4.2 Color schemes used 4 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 Fig. 1. Residues 1-61 in 1iq9A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1iq9A.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 291 Total number of residues: 16901 Smallest: 47 Largest: 61 Average length: 58.1 Alignment length: 61 Average identity: 42% Most related pair: 98% Fig. 2. Residues in 1iq9A, colored by their relative importance. Clockwise: Most unrelated pair: 16% front, back, top and bottom views. Most distant seq: 45%

Furthermore, 4% of residues show as conserved in this alignment. The alignment consists of 72% eukaryotic ( 72% vertebrata) 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 1iq9A.descr. 2.3 Residue ranking in 1iq9A The 1iq9A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1iq9A can be found in the file called 1iq9A.ranks sorted in the attachment. 2.4 Top ranking residues in 1iq9A and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1iq9A 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. 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 1iq9A, 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 15 3,17,23,24,38,39,40,42,43,53 54,56,57,59,60 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. Table 1. Clusters of top ranking residues in 1iq9A. Sulfate ion binding site. Table 2 lists the top 25% of residues at the interface with 1iq9SO4201 (sulfate ion). The following table (Table

2 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 40 C C(99)A 0.08 18/11 3.02 S-S 39 G G(98)AE 0.12 12/12 3.57 V 38 R R(75) 0.20 3/3 4.36 Q(2) L(15)I M(1) F(1)G K(1)WC 24 Y Y(75) 0.21 12/0 2.77 F(19) V(2)CLQ WI

Table 2. The top 25% of residues in 1iq9A at the interface with sulfate 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 Fig. 4. Residues in 1iq9A, at the interface with sulfate ion, colored by their type in the bracket; noc/bb: number of contacts with the ligand, with the num- relative importance. The ligand (sulfate ion) is colored green. Atoms further ber of contacts realized through backbone atoms given in the bracket; dist: than 30A˚ away from the geometric center of the ligand, as well as on the line distance of closest apporach to the ligand. ) of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1iq9A.)

Table 3. res type disruptive mutations 40 C (KER)(QHD)(FYMW)(N) 39 G (R)(K)(EH)(FYQW) 38 R (T)(D)(Y)(E) 24 Y (K)(EQR)(M)(ND)

Table 3. List of disruptive mutations for the top 25% of residues in 1iq9A, that are at the interface with sulfate ion.

Figure 4 shows residues in 1iq9A colored by their importance, at the interface with 1iq9SO4201. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1iq9A surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1iq9. It is shown in Fig. 5. The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type substitutions(%) cvg antn Fig. 5. A possible active surface on the chain 1iq9A. 53 C C(100) 0.05 S-S 54 C C(100) 0.05 S-S Table 4. continued 59 C C(100) 0.05 S-S 40 C C(99)A 0.08 S-S res type substitutions(%) cvg antn 39 G G(98)AEV 0.12 60 N N(97)D(2)S 0.10 42 C TC(97)SA(1)L 0.13 S-S continued in next column 43 P S(1)P(95)T(1) 0.15 continued in next column

3 Table 4. continued one may try replacing, R with K, or (perhaps more surprisingly), with res type substitutions(%) cvg antn V. The percentage of times the substitution appears in the alignment F(1)N is given in the immediately following bracket. No percentage is given 17 C KC(84)P(6)A(6) 0.18 S-S in the cases when it is smaller than 1%. This is meant to be a rough .(1)RL guide - due to rounding errors these percentages often do not add up 38 R R(75)Q(2)L(15)I 0.20 to 100%. M(1)F(1)GK(1)WC 24 Y Y(75)F(19)V(2)C 0.21 LQWI 3.3 Surface 56 T T(77)S(10)G(1) 0.23 To detect candidates for novel functional interfaces, first we look for K(2)P(1)R(4) residues that are solvent accessible (according to DSSP program) by 2 I(1)NA at least 10A˚ , which is roughly the area needed for one water mole- 57 D D(85)N(8)E(5)QP 0.25 cule to come in the contact with the residue. Furthermore, we require that these residues form a “cluster” of residues which have neighbor Table 4. Residues forming surface ”patch” in 1iq9A. within 5A˚ from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, the neighboring residues which are not surface accessible might be Table 5. equally important in maintaining the interaction specificity - they res type disruptive should not be automatically dropped from consideration when choo- mutations sing the set for mutagenesis. (Especially if they form a cluster with 53 C (KER)(FQMWHD)(NYLPI)(SVA) the surface residues.) 54 C (KER)(FQMWHD)(NYLPI)(SVA) 59 C (KER)(FQMWHD)(NYLPI)(SVA) 40 C (KER)(QHD)(FYMW)(N) 3.4 Number of contacts 60 N (Y)(FWH)(R)(T) Another column worth noting is denoted “noc/bb”; it tells the num- 39 G (R)(K)(EH)(FYQW) ber of contacts heavy atoms of the residue in question make across 42 C (R)(K)(E)(H) the interface, as well as how many of them are realized through the 43 P (R)(Y)(H)(K) backbone atoms (if all or most contacts are through the backbone, 17 C (E)(R)(YD)(KH) mutation presumably won’t have strong impact). Two heavy atoms 38 R (T)(D)(Y)(E) are considered to be “in contact” if their centers are closer than 5A˚ . 24 Y (K)(EQR)(M)(ND) 56 T (R)(K)(H)(FW) 57 D (R)(H)(FW)(Y) 3.5 Annotation If the residue annotation is available (either from the pdb file or Table 5. Disruptive mutations for the surface patch in 1iq9A. 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- 3.6 Mutation suggestions due is important if its “coverage” is small - that is if it belongs to 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.

4 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. 6. 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. 6. 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/

5 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. • 1iq9A.complex.pdb - coordinates of 1iq9A with all of its inter- 4.5 Citing this work acting partners The method used to rank residues and make predictions in this report • 1iq9A.etvx - ET viewer input file for 1iq9A can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1iq9A.cluster report.summary - Cluster report summary for Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 1iq9A by Importance” J. Mol. Bio. 336: 1265-82. For the original version of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 1iq9A.ranks - Ranks file in sequence order for 1iq9A tionary Trace Method Defines Binding Surfaces Common to Protein • 1iq9A.clusters - Cluster descriptions for 1iq9A Families” J. Mol. Bio. 257: 342-358. • 1iq9A.msf - the multiple sequence alignment used for the chain report maker itself is described in Mihalek I., I. Res and O. 1iq9A Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • of service for comparative analysis of proteins.” Bioinformatics 1iq9A.descr - description of sequences used in 1iq9A msf 22:1656-7. • 1iq9A.ranks sorted - full listing of residues and their ranking for 1iq9A 4.6 About report maker • 1iq9A.1iq9SO4201.if.pml - Pymol script for Figure 4 report maker was written in 2006 by Ivana Mihalek. The 1D ran- • king visualization program was written by Ivica Res.ˇ report maker 1iq9A.cbcvg - used by other 1iq9A – related pymol scripts is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston.

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