Pages 1–5 1dxj Evolutionary trace report by report maker October 31, 2010

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 1dxj): Title: Structure of the chitinase from jack Compound: Mol id: 1; molecule: class ii chitinase; chain: a; ec: 3.2.1.14 Organism, scientific name: Ensiformis; CONTENTS 1dxj contains a single unique chain 1dxjA (242 residues long). 1 Introduction 1

2 Chain 1dxjA 1 2.1 O81934 overview 1 2.2 Multiple sequence alignment for 1dxjA 1 2.3 Residue ranking in 1dxjA 1 2 CHAIN 1DXJA 2.4 Top ranking residues in 1dxjA and their position on 2.1 O81934 overview the structure 2 From SwissProt, id O81934, 95% identical to 1dxjA: 2.4.1 Clustering of residues at 25% coverage. 2 Description: Chitinase precursor. 2.4.2 Possible novel functional surfaces at 25% Organism, scientific name: Canavalia ensiformis (Jack bean) coverage. 2 (Horse bean). : Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 3 Notes on using trace results 3 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 3.1 Coverage 3 eudicotyledons; ; eurosids I; ; ; Papilionoi- 3.2 Known substitutions 3 deae; Phaseoleae; Canavalia. 3.3 Surface 4 3.4 Number of contacts 4 3.5 Annotation 4 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 1dxjA 4 Appendix 4 For the chain 1dxjA, the alignment 1dxjA.msf (attached) with 382 4.1 File formats 4 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 4 database, and fragments shorter than 75% of the query as well as 4.3 Credits 4 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 4 to this report, under the name of 1dxjA.msf. Its statistics, from the 4.3.2 CE 5 alistat program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 2-122 in 1dxjA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Fig. 2. Residues 123-243 in 1dxjA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Fig. 3. Residues in 1dxjA, colored by their relative importance. Clockwise: Format: MSF front, back, top and bottom views. Number of sequences: 382 Total number of residues: 85020 Smallest: 182 Largest: 242 Average length: 222.6 Alignment length: 242 Average identity: 46% Most related pair: 99% Most unrelated pair: 10% Most distant seq: 34%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 57% eukaryotic ( 56% plantae), and 4% prokaryotic sequences. (Descriptions of some sequences were not readily available.) The file containing the sequence descriptions can be found in the attachment, under the name 1dxjA.descr. 2.3 Residue ranking in 1dxjA The 1dxjA sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1dxjA can be found in the file called 1dxjA.ranks sorted in the attachment. Fig. 4. Residues in 1dxjA, colored according to the cluster they belong to: 2.4 Top ranking residues in 1dxjA and their position on red, followed by blue and yellow are the largest clusters (see Appendix for the structure the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 1dxjA colored by their importance: bright red and yellow indicate more conserved/important in Table 1. 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. 4 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.4 are composed of the residues listed

2 Table 1. Table 2. continued cluster size member res type substitutions(%) cvg color residues L(1)ASM red 55 12,29,30,32,35,38,42,45,46 68 E E(90)A(2)K(4)GR 0.07 57,59,60,61,62,63,64,67,68 SLQ. 69,70,110,112,113,114,115 128 G G(87)S(9)RHT.NC 0.10 117,121,123,124,125,128,135 QAV 136,139,142,149,150,152,153 168 H H(73)D.(16)L(7) 0.14 155,156,157,158,168,189,190 RTFSQ 191,194,197,198,199,202,203 198 N N(91)S(1)Y(3)T 0.15 214,218 D(1)KA.Q blue 2 164,165 32 Y Y(76)R(19)FH(1) 0.17 SLAI Table 1. Clusters of top ranking residues in 1dxjA. 199 G G(84)N(2)S(10)V 0.17 PR(1)L. 197 I I(86)L(5)V(4)KM 0.18 2.4.2 Possible novel functional surfaces at 25% coverage. One TF(1)HR.S group of residues is conserved on the 1dxjA surface, away from (or 202 E E(89)DG(1)V(2) 0.19 susbtantially larger than) other functional sites and interfaces reco- S(1)C(1)IT(1)Q gnizable in PDB entry 1dxj. It is shown in Fig. 5. The right panel .(1)PA shows (in blue) the rest of the larger cluster this surface belongs to. 156 F F(83)Y(10)KR(3) 0.21 LSE 158 M M(78)T(3)L(3) 0.21 V(4)I(1)K(1) N(6)YCA 67 H Q(14)H(77)Y(1)R 0.22 S(3)LTCA.PDF 42 F F(68)Y(29)N.LPD 0.23 70 T T(47)G(39)PDQ 0.24 S(6)N(2)AKER.IM V 125 A K(5)G(79)YC(1) 0.24 E(3)D(1)I(2) Q(1)NA(2)L(1).R Fig. 5. A possible active surface on the chain 1dxjA. The larger cluster it S belongs to is shown in blue. 136 I I(9)L(81)HV(7)W 0.24 RKYSG 31 S T(75)N(2)AS(14) 0.25 The residues belonging to this surface ”patch” are listed in Table PD(4)CHRQK 2, while Table 3 suggests possible disruptive replacements for these residues (see Section 3.6). Table 2. Residues forming surface ”patch” in 1dxjA. Table 2. res type substitutions(%) cvg 157 W W(89)F(7)Y(2)AS 0.01 Table 3. G res type disruptive 30 Y Y(92)LNW(5)KFS 0.03 mutations 69 T T(96)CPQS(1)L.Y 0.03 157 W (K)(E)(Q)(D) 155 W W(94)F(3)SCYG 0.03 30 Y (K)(EQ)(M)(R) 123 N N(97)HES.TYF 0.04 69 T (R)(K)(H)(FW) 29 F F(87)L(1).(7)G 0.05 155 W (K)(E)(Q)(D) P(2)VSIW 123 N (Y)(FWHR)(T)(E) 135 L L(72)G(24)I(1)R 0.05 29 F (K)(E)(QR)(D) AP 135 L (Y)(R)(H)(T) 117 Q Q(91)LSMP(6)K.T 0.06 117 Q (Y)(H)(FW)(T) 45 F F(95)Y(1)V(1) 0.07 45 F (K)(E)(QR)(D) continued in next column 68 E (FWH)(Y)(R)(CG) continued in next column

3 Table 3. continued 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 128 G (E)(R)(K)(D) should not be automatically dropped from consideration when choo- 168 H (E)(T)(D)(M) sing the set for mutagenesis. (Especially if they form a cluster with 198 N (Y)(FWH)(R)(T) the surface residues.) 32 Y (K)(Q)(E)(M) 199 G (ER)(K)(H)(FYWD) 3.4 Number of contacts 197 I (Y)(R)(T)(H) Another column worth noting is denoted “noc/bb”; it tells the num- 202 E (H)(R)(FW)(Y) ber of contacts heavy atoms of the residue in question make across 156 F (KE)(T)(D)(Q) the interface, as well as how many of them are realized through the 158 M (Y)(H)(R)(T) backbone atoms (if all or most contacts are through the backbone, 67 H (E)(Q)(D)(M) mutation presumably won’t have strong impact). Two heavy atoms 42 F (K)(E)(T)(R) are considered to be “in contact” if their centers are closer than 5A˚ . 70 T (R)(H)(K)(FW) 125 A (Y)(R)(KE)(H) 3.5 Annotation 136 I (R)(Y)(T)(E) 31 S (R)(K)(FW)(H) If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” appears. Annotations carried over from PDB are the following: site Table 3. Disruptive mutations for the surface patch in 1dxjA. (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 NOTES ON USING TRACE RESULTS 3.6 Mutation suggestions 3.1 Coverage Mutation suggestions are completely heuristic and based on comple- Trace results are commonly expressed in terms of coverage: the resi- mentarity with the substitutions found in the alignment. Note that due is important if its “coverage” is small - that is if it belongs to they are meant to be disruptive to the interaction of the protein some small top percentage of residues [100% is all of the residues with its ligand. The attempt is made to complement the following in a chain], according to trace. The ET results are presented in the properties: small [AV GSTC], medium [LPNQDEMIK], large form of a table, usually limited to top 25% percent of residues (or [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- to some nearby percentage), sorted by the strength of the presumed tively [KHR], or negatively [DE] charged, aromatic [WFYH], evolutionary pressure. (I.e., the smaller the coverage, the stronger the long aliphatic chain [EKRQM], OH-group possession [SDETY ], pressure on the residue.) Starting from the top of that list, mutating a and NH2 group possession [NQRK]. The suggestions are listed couple of residues should affect the protein somehow, with the exact according to how different they appear to be from the original amino effects to be determined experimentally. acid, and they are grouped in round brackets if they appear equally disruptive. From left to right, each bracketed group of amino acid 3.2 Known substitutions types resembles more strongly the original (i.e. is, presumably, less One of the table columns is “substitutions” - other amino acid types disruptive) These suggestions are tentative - they might prove disrup- seen at the same position in the alignment. These amino acid types tive to the fold rather than to the interaction. Many researcher will may be interchangeable at that position in the protein, so if one wants choose, however, the straightforward alanine mutations, especially in to affect the protein by a point mutation, they should be avoided. For the beginning stages of their investigation. 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- sely, when looking for substitutions which will not affect the protein, 4 APPENDIX one may try replacing, R with K, or (perhaps more surprisingly), with 4.1 File formats V. The percentage of times the substitution appears in the alignment Files with extension “ranks sorted” are the actual trace results. The is given in the immediately following bracket. No percentage is given fields in the table in this file: in the cases when it is smaller than 1%. This is meant to be a rough guide - due to rounding errors these percentages often do not add up • alignment# number of the position in the alignment to 100%. • residue# residue number in the PDB file 3.3 Surface • type amino acid type To detect candidates for novel functional interfaces, first we look for • rank rank of the position according to older version of ET residues that are solvent accessible (according to DSSP program) by 2 • variability has two subfields: at least 10A˚ , which is roughly the area needed for one water mole- 1. number of different amino acids appearing in in this column cule to come in the contact with the residue. Furthermore, we require of the alignment that these residues form a “cluster” of residues which have neighbor within 5A˚ from any of their heavy atoms. 2. their type

4 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- COVERAGE ble if the DSSP program finds it exposed to water by at least 10A˚ 2, which is roughly the area needed for one water molecule to come in V the contact with the residue. DSSP is copyrighted by W. Kabsch, C. 100% 50% 30% 5% Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by [email protected] November 18,2002, http://www.cmbi.kun.nl/gv/dssp/descrip.html.

4.3.4 HSSP Whenever available, report maker uses HSSP ali-

V gnment as a starting point for the analysis (sequences shorter than RELATIVE IMPORTANCE 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. Fig. 6. Coloring scheme used to color residues by their relative importance. http://swift.cmbi.kun.nl/swift/hssp/

4.3.5 LaTex The text for this report was processed using LATEX; • 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 4.3 Credits tool specifically for viewing trace results. If you are interested, please 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 of residues, the average and range of the sequence lengths, and the The viewer is self-unpacking and self-installing. Input files to be used alignment length (e.g. including gap characters). Also shown are with ETV (extension .etvx) can be found in the attachment to the some percent identities. A percent pairwise alignment identity is defi- main report. 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 sequences. The ”average percent identity”, ”most related pair”, and The method used to rank residues and make predictions in this report ”most unrelated pair” of the alignment are the average, maximum, can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant Evolution-Entropy Hybrid Methods for Ranking of Protein Residues seq” is calculated by finding the maximum pairwise identity (best by Importance” J. Mol. Bio. 336: 1265-82. For the original version relative) for all N sequences, then finding the minimum of these N of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- numbers (hence, the most outlying sequence). alistat is copyrighted tionary Trace Method Defines Binding Surfaces Common to Protein by HHMI/Washington University School of Medicine, 1992-2001, Families” J. Mol. Bio. 257: 342-358. and freely distributed under the GNU General Public License. report maker itself is described in Mihalek I., I. Res and O. 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.

5 4.6 About report maker • 1dxjA.cluster report.summary - Cluster report summary for report maker was written in 2006 by Ivana Mihalek. The 1D ran- 1dxjA king visualization program was written by Ivica Res.ˇ report maker • 1dxjA.ranks - Ranks file in sequence order for 1dxjA is copyrighted by Lichtarge Lab, Baylor College of Medicine, • 1dxjA.clusters - Cluster descriptions for 1dxjA Houston. • 1dxjA.msf - the multiple sequence alignment used for the chain 4.7 Attachments 1dxjA The following files should accompany this report: • 1dxjA.descr - description of sequences used in 1dxjA msf • 1dxjA.ranks sorted - full listing of residues and their ranking for • 1dxjA.complex.pdb - coordinates of 1dxjA with all of its inter- 1dxjA acting partners • 1dxjA.etvx - ET viewer input file for 1dxjA

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