Pages 1–7 1gyd Evolutionary trace report by report maker January 18, 2010

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 7 4.7 Attachments 7

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1gyd): Title: Structure of cellulosa alpha-l-arabinanase Compound: Mol id: 1; molecule: arabinan endo-1,5-alpha-l- arabinosidase a; chain: b; synonym: alpha-l-arabinanase, abn a, endo-1,5-alpha-l- arabinanase a; ec: 3.2.1.99; engineered: yes Organism, scientific name: Cellvibrio Japonicus; 1gyd contains a single unique chain 1gydB (315 residues long).

2 CHAIN 1GYDB 2.1 P95470 overview CONTENTS From SwissProt, id P95470, 99% identical to 1gydB: 1 Introduction 1 Description: Endo-a1,5-arabinanase (EC 3.2.1.99). Organism, scientific name: Cellvibrio japonicus. 2 Chain 1gydB 1 : ; ; Gammaproteobacteria; Pseu- 2.1 P95470 overview 1 domonadales; Pseudomonadaceae; Cellvibrio. 2.2 Multiple sequence alignment for 1gydB 1 2.3 Residue ranking in 1gydB 1 2.2 Multiple sequence alignment for 1gydB 2.4 Top ranking residues in 1gydB and their position on For the chain 1gydB, the alignment 1gydB.msf (attached) with 99 the structure 2 sequences was used. The alignment was downloaded from the HSSP 2.4.1 Clustering of residues at 25% coverage. 2 database, and fragments shorter than 75% of the query as well as 2.4.2 Possible novel functional surfaces at 25% duplicate sequences were removed. It can be found in the attachment coverage. 2 to this report, under the name of 1gydB.msf. Its statistics, from the alistat program are the following: 3 Notes on using trace results 5 3.1 Coverage 5 Format: MSF 3.2 Known substitutions 5 Number of sequences: 99 3.3 Surface 5 Total number of residues: 28097 3.4 Number of contacts 5 Smallest: 237 3.5 Annotation 5 Largest: 315 3.6 Mutation suggestions 5 Average length: 283.8 Alignment length: 315 4 Appendix 5 Average identity: 31% 4.1 File formats 5 Most related pair: 99% 4.2 Color schemes used 6 Most unrelated pair: 14% 4.3 Credits 6 Most distant seq: 31% 4.3.1 Alistat 6 4.3.2 CE 6

1 Lichtarge lab 2006 Fig. 1. Residues 33-189 in 1gydB colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Fig. 3. Residues in 1gydB, colored by their relative importance. Clockwise: front, back, top and bottom views.

Fig. 2. Residues 190-347 in 1gydB colored by their relative importance. (See Appendix, Fig.7, for the coloring scheme.)

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 8% eukaryotic ( 8% fungi), and 12% 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 1gydB.descr. 2.3 Residue ranking in 1gydB The 1gydB sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues in 1gydB can be found in the file called 1gydB.ranks sorted in the attachment. 2.4 Top ranking residues in 1gydB and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 3 shows residues in 1gydB colored by their importance: bright red and yellow indicate more conser- ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. Fig. 4. Residues in 1gydB, 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 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the corresponding Pymol script is attached. 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 in Table 1. Table 1. cluster size member color residues continued in next column

2 Table 1. continued Table 2. continued cluster size member res type substitutions(%) cvg antn color residues 175 S S(89)G(1)E(1) 0.06 red 78 37,38,39,48,49,51,53,54,57 A(3)F(1).(2) 62,64,68,94,95,96,97,105,107 T(1)N(1) 108,109,112,115,119,121,122 37 H P(16)A(8)H(71) 0.07 124,138,140,142,155,156,157 .(3)D(1) 158,159,164,167,170,171,173 142 G G(86)P(2)L(1) 0.08 174,175,176,177,179,180,185 .(4)D(1)S(2) 221,222,232,233,234,235,237 N(1)I(1)K(1) 239,241,242,248,249,251,253 241 C C(77).(5)G(1) 0.09 S-S 254,255,256,261,262,263,265 L(8)T(4)A(3) 268,271,274,276,288,290,291 F(1) 306,329,330,331 222 A G(52)A(43)P(1) 0.10 S(1).(1)N(1) Table 1. Clusters of top ranking residues in 1gydB. 288 G G(77)A(7)E(2) 0.10 S(10).(3) 155 N N(84)G(4)E(4) 0.11 2.4.2 Possible novel functional surfaces at 25% coverage. One W(1)Q(2)R(1) group of residues is conserved on the 1gydB surface, away from (or D(1).(1)Y(1) susbtantially larger than) other functional sites and interfaces reco- 239 G G(69)D(23)S(1) 0.11 gnizable in PDB entry 1gyd. It is shown in Fig. 5. The right panel T(1)N(3)P(1) shows (in blue) the rest of the larger cluster this surface belongs to. R(1) 242 C C(77).(4)G(2) 0.12 S-S M(3)D(5)S(1) Q(1)W(2)N(1) A(3) 248 T T(48)G(10)P(2) 0.12 A(1)E(28)D(5) K(1)Q(1)S(2) R(1) 112 S S(78)V(2)Y(1) 0.13 R(4)C(4)A(2) H(1)N(3)T(2) G(1)I(1) 53 T A(12)T(67)G(7) 0.14 Fig. 5. A possible active surface on the chain 1gydB. The larger cluster it V(3).(5)S(2) belongs to is shown in blue. F(1)Y(2) 105 F Y(73)F(20)T(1) 0.14 W(1)V(3).(1) The residues belonging to this surface ”patch” are listed in Table 140 D D(79)Q(2)G(3) 0.15 2, while Table 3 suggests possible disruptive replacements for these A(2)K(1)E(2) residues (see Section 3.6). .(4)P(1)L(1) Table 2. V(2)N(2) 138 W F(16)W(66)Y(5) 0.16 res type substitutions(%) cvg antn R(2)H(1)N(1) 158 D D(100) 0.01 .(4)V(2)P(1) 249 Y Y(94)H(3)L(1) 0.01 S(1) E(1) 291 H H(56)G(29)P(2) 0.17 38 D D(98)P(1) 0.02 R(1).(3)Q(1) 96 P P(98)A(1) 0.02 V(4)M(1)A(1) 221 E E(97)Q(1).(1) 0.03 I(1) 94 W W(93)F(2)G(1) 0.04 54 G T(14)E(2)G(63) 0.18 L(1)Q(1)A(1) A(4)S(4)D(3) 157 I I(93)V(3)F(1) 0.04 L(2) continued in next column continued in next column

3 Table 2. continued Table 3. continued res type substitutions(%) cvg antn res type disruptive M(1)Q(2)H(4) mutations L(2) 291 H (E)(TD)(Q)(S) 159 P Q(4)P(68)A(18) 0.19 54 G (R)(K)(H)(E) G(9) 159 P (Y)(R)(H)(TE) 176 F F(62)Y(26)W(6) 0.20 176 F (K)(E)(QR)(T) N(1)D(2)T(1) 177 W (E)(K)(D)(T) P(1) 97 D (R)(H)(FW)(K) 177 W R(7)H(5)W(54) 0.21 115 G (R)(K)(H)(E) .(4)F(14)G(4) 106 Y (K)(Q)(E)(R) S(5)N(3)I(1) L(2) Table 3. Disruptive mutations for the surface patch in 1gydB. 97 D D(69)V(1)N(2) 0.23 T(5)E(8)F(1) Another group of surface residues is shown in Fig.6. The right panel H(3)Y(2)A(1) shows (in blue) the rest of the larger cluster this surface belongs to. S(1)M(2)Q(4) 115 G G(78)D(5)S(3) 0.23 E(1)A(3)K(2) N(2).(1)Y(1) L(1)I(1)P(1) 106 Y V(13)Y(55)W(6) 0.25 H(9)R(5)S(1) F(1)L(4).(2) M(1)I(2)

Table 2. Residues forming surface ”patch” in 1gydB.

Table 3. res type disruptive Fig. 6. Another possible active surface on the chain 1gydB. The larger cluster mutations it belongs to is shown in blue. 158 D (R)(FWH)(KYVCAG)(TQM) 249 Y (K)(Q)(MR)(VA) The residues belonging to this surface ”patch” are listed in Table 38 D (R)(H)(FYW)(KCG) 4, while Table 5 suggests possible disruptive replacements for these 96 P (YR)(H)(TKE)(SQCDG) residues (see Section 3.6). 221 E (FWH)(YVCAG)(TR)(S) 94 W (E)(K)(D)(T) Table 4. 157 I (R)(Y)(T)(KEH) res type substitutions(%) cvg 175 S (R)(K)(H)(FQW) 233 Y Y(97)W(1)F(1) 0.02 37 H (E)(Q)(T)(K) 255 R R(97)Y(1)T(1) 0.03 142 G (R)(H)(KE)(FW) 256 S S(93)A(5)T(1) 0.04 241 C (KR)(E)(Q)(H) 261 G G(97)N(1)E(1) 0.04 222 A (R)(Y)(KE)(H) 232 Y Y(93)F(3)H(2) 0.05 288 G (R)(K)(H)(FW) W(1) 155 N (Y)(FTWH)(VA)(CG) 265 D D(84)T(2)G(3) 0.07 239 G (R)(K)(E)(H) N(4)K(4)H(1) 242 C (R)(K)(E)(H) Y(1) 248 T (R)(FWH)(K)(M) 330 W W(91).(5)F(1) 0.08 112 S (K)(R)(Q)(M) K(1)L(1) 53 T (K)(R)(Q)(M) 262 P P(81)N(6)H(1) 0.10 105 F (K)(E)(Q)(D) G(7)L(1)D(3) 140 D (R)(H)(FYW)(CG) 253 V V(83)Y(8)I(2) 0.11 138 W (E)(K)(D)(Q) A(2)M(2)S(1) continued in next column Q(1) continued in next column

4 Table 4. continued Table 5. continued res type substitutions(%) cvg res type disruptive 167 G G(86)A(1)T(1) 0.12 mutations S(5)Q(2)D(2) 170 W (E)(K)(D)(Q) E(1)K(1) 185 L (R)(Y)(H)(K) 268 G G(87)A(1)Q(2) 0.16 329 G (R)(FW)(K)(H) E(1)H(1)V(1) 274 G (R)(K)(H)(E) .(1)N(3)L(1) 276 G (KR)(E)(FW)(M) C(1) 231 Y (K)(QM)(VA)(R) 164 D D(78)E(7)T(3) 0.17 Q(1)I(3)G(1) Table 5. Disruptive mutations for the surface patch in 1gydB. A(4)S(1)V(1) 331 P P(85).(6)F(5) 0.17 E(2)L(1) 170 W Y(42)F(4)W(44) 0.19 3 NOTES ON USING TRACE RESULTS H(2)K(1)A(1) 3.1 Coverage V(3).(1)I(1) Trace results are commonly expressed in terms of coverage: the resi- 185 L S(5)L(69)T(3) 0.22 due is important if its “coverage” is small - that is if it belongs to M(14)V(2)A(1) some small top percentage of residues [100% is all of the residues I(5) in a chain], according to trace. The ET results are presented in the 329 G G(83).(4)D(6) 0.22 form of a table, usually limited to top 25% percent of residues (or E(1)S(1)N(1) to some nearby percentage), sorted by the strength of the presumed H(1)K(1)P(1) evolutionary pressure. (I.e., the smaller the coverage, the stronger the 274 G N(15)E(2)G(60) 0.24 pressure on the residue.) Starting from the top of that list, mutating a K(1)S(6)T(4) couple of residues should affect the protein somehow, with the exact C(4).(4)I(2) effects to be determined experimentally. V(1) 276 G H(10)S(2)G(68) 0.24 3.2 Known substitutions D(2)Y(3)V(1) One of the table columns is “substitutions” - other amino acid types E(1)N(4)T(1) seen at the same position in the alignment. These amino acid types W(1).(3)R(1) may be interchangeable at that position in the protein, so if one wants K(2) to affect the protein by a point mutation, they should be avoided. For 231 Y Y(72)F(9)W(11) 0.25 example if the substitutions are “RVK” and the original protein has H(1).(1)D(1) an R at that position, it is advisable to try anything, but RVK. Conver- Q(1)E(1)K(2) sely, when looking for substitutions which will not affect the protein, one may try replacing, R with K, or (perhaps more surprisingly), with Table 4. Residues forming surface ”patch” in 1gydB. V. The percentage of times the substitution appears in the alignment is given in the immediately following bracket. No percentage is given in the cases when it is smaller than 1%. This is meant to be a rough Table 5. guide - due to rounding errors these percentages often do not add up res type disruptive to 100%. mutations 233 Y (K)(Q)(E)(M) 3.3 Surface 255 R (D)(LPI)(E)(VA) To detect candidates for novel functional interfaces, first we look for 256 S (KR)(QH)(FMW)(E) residues that are solvent accessible (according to DSSP program) by 2 261 G (R)(FWH)(K)(E) at least 10A˚ , which is roughly the area needed for one water mole- 232 Y (K)(Q)(E)(M) cule to come in the contact with the residue. Furthermore, we require 265 D (R)(FW)(H)(K) that these residues form a “cluster” of residues which have neighbor 330 W (E)(T)(K)(D) within 5A˚ from any of their heavy atoms. 262 P (R)(Y)(H)(T) Note, however, that, if our picture of protein evolution is correct, 253 V (R)(Y)(K)(E) the neighboring residues which are not surface accessible might be 167 G (R)(FWH)(K)(E) equally important in maintaining the interaction specificity - they 268 G (R)(E)(K)(H) should not be automatically dropped from consideration when choo- 164 D (R)(H)(FW)(Y) sing the set for mutagenesis. (Especially if they form a cluster with 331 P (R)(Y)(T)(H) the surface residues.) continued in next column 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

5 the interface, as well as how many of them are realized through the backbone atoms (if all or most contacts are through the backbone, mutation presumably won’t have strong impact). Two heavy atoms are considered to be “in contact” if their centers are closer than 5A˚ . 3.5 Annotation COVERAGE

If the residue annotation is available (either from the pdb file or V from other sources), another column, with the header “annotation” 100% 50% 30% 5% 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 bond forming residue), and sb (for salt bridge forming residue).

3.6 Mutation suggestions V

Mutation suggestions are completely heuristic and based on comple- RELATIVE IMPORTANCE mentarity with the substitutions found in the alignment. Note that they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following Fig. 7. Coloring scheme used to color residues by their relative importance. properties: small [AV GSTC], medium [LPNQDEMIK], large [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- tively [KHR], or negatively [DE] charged, aromatic [WFYH], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDETY ], quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, and NH2 group possession [NQRK]. The suggestions are listed bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, according to how different they appear to be from the original amino DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, acid, and they are grouped in round brackets if they appear equally tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. disruptive. From left to right, each bracketed group of amino acid The colors used to distinguish the residues by the estimated types resembles more strongly the original (i.e. is, presumably, less evolutionary pressure they experience can be seen in Fig. 7. disruptive) These suggestions are tentative - they might prove disrup- tive to the fold rather than to the interaction. Many researcher will 4.3 Credits choose, however, the straightforward alanine mutations, especially in 4.3.1 Alistat alistat reads a multiple sequence alignment from the the beginning stages of their investigation. file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number 4 APPENDIX of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are 4.1 File formats some percent identities. A percent pairwise alignment identity is defi- Files with extension “ranks sorted” are the actual trace results. The ned as (idents / MIN(len1, len2)) where idents is the number of fields in the table in this file: exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and • alignment# number of the position in the alignment ”most unrelated pair” of the alignment are the average, maximum, • residue# residue number in the PDB file and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant • type amino acid type seq” is calculated by finding the maximum pairwise identity (best relative) for all N sequences, then finding the minimum of these N • rank rank of the position according to older version of ET numbers (hence, the most outlying sequence). alistat is copyrighted • variability has two subfields: by HHMI/Washington University School of Medicine, 1992-2001, 1. number of different amino acids appearing in in this column and freely distributed under the GNU General Public License. of the alignment 4.3.2 CE To map ligand binding sites from different 2. their type source structures, report maker uses the CE program: • rho ET score - the smaller this value, the lesser variability of http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) this position across the branches of the tree (and, presumably, ”Protein structure alignment by incremental combinatorial extension the greater the importance for the protein) (CE) of the optimal path . Protein Engineering 11(9) 739-747. • cvg coverage - percentage of the residues on the structure which 4.3.3 DSSP In this work a residue is considered solvent accessi- have this rho or smaller ble if the DSSP program finds it exposed to water by at least 10A˚ 2, • gaps percentage of gaps in this column 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. 4.2 Color schemes used Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version The following color scheme is used in figures with residues colored by [email protected] November 18,2002, by cluster size: black is a single-residue cluster; clusters composed of more than one residue colored according to this hierarchy (ordered http://www.cmbi.kun.nl/gv/dssp/descrip.html.

6 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 4.5 Citing this work gnment as a starting point for the analysis (sequences shorter than The method used to rank residues and make predictions in this report 75% of the query are taken out, however); R. Schneider, A. de can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of Daruvar, and C. Sander. ”The HSSP database of protein structure- Evolution-Entropy Hybrid Methods for Ranking of Protein Residues sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 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- http://swift.cmbi.kun.nl/swift/hssp/ tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. 4.3.5 LaTex The text for this report was processed using LAT X; E report maker itself is described in Mihalek I., I. Res and O. Leslie Lamport, “LaTeX: A Document Preparation System Addison- Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type Wesley,” Reading, Mass. (1986). of service for comparative analysis of proteins.” Bioinformatics 4.3.6 Muscle When making alignments “from scratch”, report 22:1656-7. maker uses Muscle alignment program: Edgar, Robert C. (2004), 4.6 About report maker ”MUSCLE: multiple sequence alignment with high accuracy and high throughput.” Nucleic Acids Research 32(5), 1792-97. report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker http://www.drive5.com/muscle/ is copyrighted by Lichtarge Lab, Baylor College of Medicine, Houston. 4.3.7 Pymol The figures in this report were produced using 4.7 Attachments Pymol. The scripts can be found in the attachment. Pymol is an open-source application copyrighted by DeLano Scien- The following files should accompany this report: tific LLC (2005). For more information about Pymol see • 1gydB.complex.pdb - coordinates of 1gydB with all of its http://pymol.sourceforge.net/ . (Note for Windows interacting partners users: the attached package needs to be unzipped for Pymol to read • the scripts and launch the viewer.) 1gydB.etvx - ET viewer input file for 1gydB • 1gydB.cluster report.summary - Cluster report summary for 4.4 Note about ET Viewer 1gydB Dan Morgan from the Lichtarge lab has developed a visualization • 1gydB.ranks - Ranks file in sequence order for 1gydB tool specifically for viewing trace results. If you are interested, please • 1gydB.clusters - Cluster descriptions for 1gydB visit: • 1gydB.msf - the multiple sequence alignment used for the chain http://mammoth.bcm.tmc.edu/traceview/ 1gydB • 1gydB.descr - description of sequences used in 1gydB msf The viewer is self-unpacking and self-installing. Input files to be used • 1gydB.ranks sorted - full listing of residues and their ranking with ETV (extension .etvx) can be found in the attachment to the for 1gydB main report.

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