Pages 1–5 2zib Evolutionary trace report by report maker September 10, 2008

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 2zib): Title: Crystal structure analysis of calcium-independent type ii antifreeze protein Compound: Mol id: 1; molecule: type ii antifreeze protein; chain: a; engineered: yes Organism, scientific name: Brachyopsis Rostratus; 2zib contains a single unique chain 2zibA (130 residues long). CONTENTS

1 Introduction 1 2 CHAIN 2ZIBA 2.1 P05140 overview 2 Chain 2zibA 1 From SwissProt, id P05140, 68% identical to 2zibA: 2.1 P05140 overview 1 Description: Type II antifreeze protein precursor (AFP). 2.2 Multiple sequence alignment for 2zibA 1 Organism, scientific name: (). 2.3 Residue ranking in 2zibA 1 : Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 2.4 Top ranking residues in 2zibA and their position on Euteleostomi; ; Neopterygii; Teleostei; Euteleostei; the structure 1 Neoteleostei; Acanthomorpha; Acanthopterygii; Percomorpha; Scor- 2.4.1 Clustering of residues at 25% coverage. 2 paeniformes; Cottoidei; Hemitripteridae; Hemitripterus. 2.4.2 Overlap with known functional surfaces at Function: Antifreeze proteins lower the blood freezing point. 25% coverage. 2 Subcellular location: Secreted. 2.4.3 Possible novel functional surfaces at 25% Similarity: Contains 1 C-type lectin domain. coverage. 3 About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 3 collaboration between the Swiss Institute of Bioinformatics and the EMBL outstation - the European Bioinformatics Institute. There are 3.1 Coverage 3 no restrictions on its use as long as its content is in no way modified 3.2 Known substitutions 4 and this statement is not removed. 3.3 Surface 4 3.4 Number of contacts 4 2.2 Multiple sequence alignment for 2zibA 3.5 Annotation 4 3.6 Mutation suggestions 4 For the chain 2zibA, the alignment 2zibA.msf (attached) with 28 sequences was used. The alignment was assembled through combi- 4 Appendix 4 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 4 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 5 report, under the name of 2zibA.msf. Its statistics, from the alistat 4.3 Credits 5 program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 4-133 in 2zibA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Format: MSF Number of sequences: 28 Total number of residues: 3579 Smallest: 120 Largest: 130 Average length: 127.8 Alignment length: 130 Average identity: 36% Most related pair: 98% Most unrelated pair: 21% Most distant seq: 33% Fig. 2. Residues in 2zibA, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, 5% of residues show as conserved in this alignment. The alignment consists of 96% eukaryotic ( 85% 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 2zibA.descr. 2.3 Residue ranking in 2zibA The 2zibA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2zibA can be found in the file called 2zibA.ranks sorted in the attachment. 2.4 Top ranking residues in 2zibA 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 2zibA 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 in Table 1. Fig. 3. Residues in 2zibA, colored according to the cluster they belong to: Table 1. red, followed by blue and yellow are the largest clusters (see Appendix for cluster size member the coloring scheme). Clockwise: front, back, top and bottom views. The color residues corresponding Pymol script is attached. red 33 10,14,17,21,22,31,34,35,38 44,45,47,53,56,57,66,67,68 69,78,80,82,90,91,96,104,115 116,117,120,128,129,130 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 2zibA. Sulfate ion binding site. Table 2 lists the top 25% of residues at the interface with 2zibASO4501 (sulfate ion). The following table

2 (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 82 D D(85) 0.18 1/1 4.75 E(7) R(3) N(3) 44 H H(78) 0.21 15/0 2.66 Q(7) V(3) N(7) S(3)

Table 2. The top 25% of residues in 2zibA 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 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. )

Fig. 4. Residues in 2zibA, at the interface with sulfate ion, colored by their Table 3. relative importance. The ligand (sulfate ion) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line res type disruptive of sight to the ligand were removed. (See Appendix for the coloring scheme mutations for the protein chain 2zibA.) 82 D (FW)(HR)(Y)(VCAG) 44 H (E)(TD)(M)(Q)

Table 3. List of disruptive mutations for the top 25% of residues in 2zibA, that are at the interface with sulfate ion.

Figure 4 shows residues in 2zibA colored by their importance, at the interface with 2zibASO4501. 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 2zibA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 2zib. It is shown in Fig. 5. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. Fig. 5. A possible active surface on the chain 2zibA. The larger cluster it The residues belonging to this surface ”patch” are listed in Table belongs to is shown in blue. 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. continued Table 4. res type substitutions(%) cvg antn res type substitutions(%) cvg antn N(3) 80 W W(100) 0.05 78 W F(50)W(46)Y(3) 0.19 120 C C(92)L(7) 0.07 S-S 35 E E(78)Q(10)K(7) 0.20 91 W W(92)I(7) 0.08 S(3) 14 W W(96)Y(3) 0.09 44 H H(78)Q(7)V(3) 0.21 31 W W(96)Y(3) 0.10 N(7)S(3) 96 P P(85).(7)F(7) 0.12 116 D N(71)D(10)A(3) 0.21 10 C C(89)V(7)I(3) 0.15 S-S V(7)R(7) 129 A K(64)Q(25)E(3) 0.17 117 D D(67)S(3)T(7) 0.22 A(7) P(7)N(14) 82 D D(85)E(7)R(3) 0.18 90 T N(60)F(10)A(7) 0.23 continued in next column T(7)Q(3)I(7) continued in next column

3 Table 4. continued guide - due to rounding errors these percentages often do not add up res type substitutions(%) cvg antn to 100%. D(3) 130 K K(64)R(10)E(7) 0.25 3.3 Surface I(10)M(7) To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 Table 4. Residues forming surface ”patch” in 2zibA. 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 A˚ Table 5. within 5 from any of their heavy atoms. Note, however, that, if our picture of protein evolution is correct, res type disruptive the neighboring residues which are not surface accessible might be mutations equally important in maintaining the interaction specificity - they 80 W (KE)(TQD)(SNCRG)(M) should not be automatically dropped from consideration when choo- 120 C (R)(KE)(H)(FYQWD) sing the set for mutagenesis. (Especially if they form a cluster with 91 W (KE)(T)(QDR)(SCG) the surface residues.) 14 W (K)(E)(Q)(D) 31 W (K)(E)(Q)(D) 3.4 Number of contacts 96 P (R)(Y)(T)(H) Another column worth noting is denoted “noc/bb”; it tells the num- 10 C (R)(KE)(H)(Y) ber of contacts heavy atoms of the residue in question make across 129 A (Y)(H)(R)(E) the interface, as well as how many of them are realized through the 82 D (FW)(HR)(Y)(VCAG) backbone atoms (if all or most contacts are through the backbone, 78 W (K)(E)(Q)(D) mutation presumably won’t have strong impact). Two heavy atoms 35 E (FW)(H)(Y)(VCAG) are considered to be “in contact” if their centers are closer than 5A˚ . 44 H (E)(TD)(M)(Q) 116 D (R)(Y)(H)(FW) 3.5 Annotation 117 D (R)(H)(FW)(K) If the residue annotation is available (either from the pdb file or 90 T (R)(K)(H)(FW) from other sources), another column, with the header “annotation” 130 K (Y)(T)(FW)(CG) 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 2zibA. 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 GST C], medium [LP NQDEMIK], large some small top percentage of residues [100% is all of the residues [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [W F Y H], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDET Y ], 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 4 APPENDIX 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- 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

4 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. COVERAGE 4.3.2 CE To map ligand binding sites from different V source structures, report maker uses the CE program: 100% 50% 30% 5% http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) ”Protein structure alignment by incremental combinatorial extension (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 ble if the DSSP program finds it exposed to water by at least 10A˚ , V which is roughly the area needed for one water molecule to come in RELATIVE IMPORTANCE 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, Fig. 6. Coloring scheme used to color residues by their relative importance. http://www.cmbi.kun.nl/gv/dssp/descrip.html.

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 4.3.5 LaTex The text for this report was processed using LAT X; • E rho ET score - the smaller this value, the lesser variability of Leslie Lamport, “LaTeX: A Document Preparation System Addison- this position across the branches of the tree (and, presumably, Wesley,” Reading, Mass. (1986). the greater the importance for the protein) 4.3.6 Muscle When making alignments “from scratch”, report • cvg coverage - percentage of the residues on the structure which maker uses Muscle alignment program: Edgar, Robert C. (2004), have this rho or smaller ”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. 6. 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 http://mammoth.bcm.tmc.edu/traceview/ file and shows a number of simple statistics about it. These stati- 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

5 of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- • 2zibA.complex.pdb - coordinates of 2zibA with all of its inter- tionary Trace Method Defines Binding Surfaces Common to Protein acting partners Families” J. Mol. Bio. 257: 342-358. • 2zibA.etvx - ET viewer input file for 2zibA report maker itself is described in Mihalek I., I. Res and O. • 2zibA.cluster report.summary - Cluster report summary for Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 2zibA of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 2zibA.ranks - Ranks file in sequence order for 2zibA • 2zibA.clusters - Cluster descriptions for 2zibA • 4.6 About report maker 2zibA.msf - the multiple sequence alignment used for the chain 2zibA report maker was written in 2006 by Ivana Mihalek. The 1D ran- • king visualization program was written by Ivica Res.ˇ report maker 2zibA.descr - description of sequences used in 2zibA msf is copyrighted by Lichtarge Lab, Baylor College of Medicine, • 2zibA.ranks sorted - full listing of residues and their ranking for Houston. 2zibA • 2zibA.2zibASO4501.if.pml - Pymol script for Figure 4 • 4.7 Attachments 2zibA.cbcvg - used by other 2zibA – related pymol scripts The following files should accompany this report:

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