Pages 1–5 1gp4 Evolutionary trace report by report maker August 20, 2009

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 1gp4): Title: Anthocyanidin synthase from (selenome- thionine substituted) Compound: Mol id: 1; molecule: anthocyanidin synthase; chain: a; synonym: dioxygenase; engineered: yes Organism, scientific name: Arabidopsis Thaliana; 1gp4 contains a single unique chain 1gp4A (346 residues long).

2 CHAIN 1GP4A 2.1 Q96323 overview CONTENTS From SwissProt, id Q96323, 94% identical to 1gp4A: 1 Introduction 1 Description: Leucoanthocyanidin dioxygenase (EC 1.14.11.19) (LDOX) (Leucocyanidin ) (Leucoanthocyanidin hydro- 2 Chain 1gp4A 1 xylase) (Anthocyanidin synthase) (ANS). 2.1 Q96323 overview 1 Organism, scientific name: Arabidopsis thaliana (Mouse-ear cress). 2.2 Multiple sequence alignment for 1gp4A 1 Taxonomy: Eukaryota; Viridiplantae; Streptophyta; Embryophyta; 2.3 Residue ranking in 1gp4A 1 Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core 2.4 Top ranking residues in 1gp4A and their position on eudicotyledons; rosids; eurosids II; Brassicales; Brassicaceae; Ara- the structure 1 bidopsis. 2.4.1 Clustering of residues at 25% coverage. 2 Function: Oxidation of into anthocyanidins. 2.4.2 Overlap with known functional surfaces at Catalytic activity: Leucocyanidin + 2-oxoglutarate + O(2) = cis- and 25% coverage. 2 trans-dihydroquercetins + succinate + CO(2). : Binds 1 iron ion and 1 ascorbate molecule per subunit. 3 Notes on using trace results 3 Pathway: Flavonoid synthesis; anthocyanidins biosynthesis. 3.1 Coverage 3 Similarity: Belongs to the iron/ascorbate-dependent 3.2 Known substitutions 4 family. 3.3 Surface 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.4 Number of contacts 4 collaboration between the Swiss Institute of Bioinformatics and the 3.5 Annotation 4 EMBL outstation - the European Bioinformatics Institute. There are 3.6 Mutation suggestions 4 no restrictions on its use as long as its content is in no way modified and this statement is not removed. 4 Appendix 4 4.1 File formats 4 2.2 Multiple sequence alignment for 1gp4A 4.2 Color schemes used 4 For the chain 1gp4A, the alignment 1gp4A.msf (attached) with 611 4.3 Credits 5 sequences was used. The alignment was downloaded from the HSSP 4.3.1 Alistat 5 database, and fragments shorter than 75% of the query as well as 4.3.2 CE 5 duplicate sequences were removed. It can be found in the attachment

1 Lichtarge lab 2006 2.4 Top ranking residues in 1gp4A 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 1gp4A 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. 1. Residues 2-174 in 1gp4A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Fig. 2. Residues 175-347 in 1gp4A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

to this report, under the name of 1gp4A.msf. Its statistics, from the alistat program are the following: Fig. 3. Residues in 1gp4A, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 611 Total number of residues: 191106 Smallest: 260 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Largest: 346 top 25% of all residues, this time colored according to clusters they Average length: 312.8 belong to. The clusters in Fig.4 are composed of the residues listed Alignment length: 346 in Table 1. Average identity: 34% Table 1. Most related pair: 99% cluster size member Most unrelated pair: 14% color residues Most distant seq: 32% red 86 48,51,73,74,77,78,79,80,81 84,85,86,87,102,103,105,106 110,138,139,141,172,179,187 Furthermore, <1% of residues show as conserved in this ali- 188,190,191,192,193,198,213 gnment. 215,216,217,218,220,221,223 The alignment consists of 48% eukaryotic ( 48% plantae) 225,226,227,228,231,232,233 sequences. (Descriptions of some sequences were not readily availa- 234,238,239,241,242,243,244 ble.) The file containing the sequence descriptions can be found in 246,248,249,250,251,257,260 the attachment, under the name 1gp4A.descr. 262,266,267,268,269,270,272 273,274,276,277,279,280,281 2.3 Residue ranking in 1gp4A 283,285,286,288,289,290,292 293,298,300,302,304,307 The 1gp4A sequence is shown in Figs. 1–2, with each residue colored according to its estimated importance. The full listing of residues continued in next column in 1gp4A can be found in the file called 1gp4A.ranks sorted in the attachment.

2 Table 2. continued res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 300 S S(96)C 0.06 10/1 2.77 T(1).VY N 215 N S(23)Y 0.07 17/0 3.18 site N(64) H(4)Q T(3) A(2)LIC EXRV 241 I L(85) 0.09 6/0 4.17 I(11) V(2)F.Q M 304 F F(92) 0.11 9/0 3.30 L(4)VT .(1)IAC SGYPH 213 K K(42) 0.14 4/0 4.31 R(27) Fig. 4. Residues in 1gp4A, colored according to the cluster they belong to: V(1) red, followed by blue and yellow are the largest clusters (see Appendix for .(14)I the coloring scheme). Clockwise: front, back, top and bottom views. The Q(2) corresponding Pymol script is attached. A(3) N(1) L(1)GES Table 1. continued T(1) cluster size member H(1)F color residues 302 A A(78) 0.18 2/0 3.62 P(11)T Table 1. Clusters of top ranking residues in 1gp4A. V(4) G(2)SL. YD 2.4.2 Overlap with known functional surfaces at 25% coverage. The name of the ligand is composed of the source PDB identifier Table 2. and the heteroatom name used in that file. The top 25% of residues in 1gp4A at the interface with 2- oxyglutaric acid.(Field names: res: residue number in the PDB entry; type: 2-oxyglutaric acid . Table 2 lists the top 25% of resi- amino acid type; substs: substitutions seen in the alignment; with the percen- dues at the interface with 1gp4AAKG370 (2-oxyglutaric acid). The tage of each type in the bracket; noc/bb: number of contacts with the ligand, following table (Table 3) suggests possible disruptive replacements with the number of contacts realized through backbone atoms given in the for these residues (see Section 3.6). bracket; dist: distance of closest apporach to the ligand. ) Table 2. res type subst’s cvg noc/ dist antn Table 3. (%) bb (A˚ ) res type disruptive 234 D D(99).E 0.00 5/0 3.77 site mutations 249 L L(99)T. 0.01 6/1 4.14 234 D (R)(FW)(H)(VCAG) F 249 L (R)(Y)(KH)(TE) 232 H H(99)T. 0.02 11/0 3.25 site 232 H (E)(MD)(Q)(TLPI) R 217 Y (K)(R)(Q)(E) 217 Y Y(98)LM 0.03 13/0 2.83 site 288 H (E)(T)(D)(M) FQ 298 R (D)(TY)(E)(S) 288 H H(98)K. 0.04 6/0 3.54 site 300 S (KR)(M)(QH)(FW) ASLQ 215 N (Y)(H)(FW)(R) 298 R R(98).X 0.06 9/0 2.82 site 241 I (Y)(R)(T)(H) AGIM continued in next column continued in next column

3 Table 3. continued 3.2 Known substitutions res type disruptive One of the table columns is “substitutions” - other amino acid types mutations seen at the same position in the alignment. These amino acid types 304 F (K)(E)(Q)(R) may be interchangeable at that position in the protein, so if one wants 213 K (Y)(FW)(T)(CG) to affect the protein by a point mutation, they should be avoided. For 302 A (R)(K)(E)(Y) 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- Table 3. List of disruptive mutations for the top 25% of residues in sely, when looking for substitutions which will not affect the protein, 1gp4A, that are at the interface with 2-oxyglutaric acid. one may try replacing, R with K, or (perhaps more surprisingly), with 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 guide - due to rounding errors these percentages often do not add up to 100%. 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by 2 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 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 equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- sing the set for mutagenesis. (Especially if they form a cluster with the surface residues.) 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 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 Fig. 5. Residues in 1gp4A, at the interface with 2-oxyglutaric acid, colored are considered to be “in contact” if their centers are closer than 5A˚ . by their relative importance. The ligand (2-oxyglutaric acid) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well 3.5 Annotation as on the line of sight to the ligand were removed. (See Appendix for the If the residue annotation is available (either from the pdb file or coloring scheme for the protein chain 1gp4A.) 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 Figure 5 shows residues in 1gp4A colored by their importance, at the bond forming residue), hb (hydrogen bond forming residue, jb (james interface with 1gp4AAKG370. 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- mentarity with the substitutions found in the alignment. Note that 3.1 Coverage 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 GSTC], medium [LPNQDEMIK], large some small top percentage of residues [100% is all of the residues [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- in a chain], according to trace. The ET results are presented in the tively [KHR], or negatively [DE] charged, aromatic [WFYH], form of a table, usually limited to top 25% percent of residues (or long aliphatic chain [EKRQM], OH-group possession [SDETY ], 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

4 4.3 Credits 4.3.1 Alistat alistat reads a multiple sequence alignment from the file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number COVERAGE of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are V some percent identities. A percent pairwise alignment identity is defi- 100% 50% 30% 5% ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two sequences. The ”average percent identity”, ”most related pair”, and ”most unrelated pair” of the alignment are the average, maximum, and minimum of all (N)(N-1)/2 pairs, respectively. The ”most distant seq” is calculated by finding the maximum pairwise identity (best V relative) for all N sequences, then finding the minimum of these N RELATIVE IMPORTANCE 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. Fig. 6. Coloring scheme used to color residues by their relative importance. 4.3.2 CE To map ligand binding sites from different source structures, report maker uses the CE program: disruptive) These suggestions are tentative - they might prove disrup- http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) tive to the fold rather than to the interaction. Many researcher will ”Protein structure alignment by incremental combinatorial extension choose, however, the straightforward alanine mutations, especially in (CE) of the optimal path . Protein Engineering 11(9) 739-747. the beginning stages of their investigation. 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 • type amino acid type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- 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 2. their type http://swift.cmbi.kun.nl/swift/hssp/ • rho ET score - the smaller this value, the lesser variability of this position across the branches of the tree (and, presumably, 4.3.5 LaTex The text for this report was processed using LATEX; the greater the importance for the protein) Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which have this rho or smaller 4.3.6 Muscle When making alignments “from scratch”, report • gaps percentage of gaps in this column maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and 4.2 Color schemes used high throughput.” Nucleic Acids Research 32(5), 1792-97. The following color scheme is used in figures with residues colored by cluster size: black is a single-residue cluster; clusters composed of http://www.drive5.com/muscle/ more than one residue colored according to this hierarchy (ordered by descending size): red, blue, yellow, green, purple, azure, tur- 4.3.7 Pymol The figures in this report were produced using quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, Pymol. The scripts can be found in the attachment. Pymol bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, is an open-source application copyrighted by DeLano Scien- DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tific LLC (2005). For more information about Pymol see tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. http://pymol.sourceforge.net/. (Note for Windows The colors used to distinguish the residues by the estimated users: the attached package needs to be unzipped for Pymol to read evolutionary pressure they experience can be seen in Fig. 6. the scripts and launch the viewer.)

5 4.4 Note about ET Viewer is copyrighted by Lichtarge Lab, Baylor College of Medicine, Dan Morgan from the Lichtarge lab has developed a visualization Houston. tool specifically for viewing trace results. If you are interested, please 4.7 Attachments visit: The following files should accompany this report: http://mammoth.bcm.tmc.edu/traceview/ • 1gp4A.complex.pdb - coordinates of 1gp4A with all of its The viewer is self-unpacking and self-installing. Input files to be used interacting partners with ETV (extension .etvx) can be found in the attachment to the • 1gp4A.etvx - ET viewer input file for 1gp4A main report. • 1gp4A.cluster report.summary - Cluster report summary for 4.5 Citing this work 1gp4A The method used to rank residues and make predictions in this report • 1gp4A.ranks - Ranks file in sequence order for 1gp4A can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1gp4A.clusters - Cluster descriptions for 1gp4A Evolution-Entropy Hybrid Methods for Ranking of Protein Residues • 1gp4A.msf - the multiple sequence alignment used for the chain by Importance” J. Mol. Bio. 336: 1265-82. For the original version 1gp4A of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- tionary Trace Method Defines Binding Surfaces Common to Protein • 1gp4A.descr - description of sequences used in 1gp4A msf Families” J. Mol. Bio. 257: 342-358. • 1gp4A.ranks sorted - full listing of residues and their ranking report maker itself is described in Mihalek I., I. Res and O. for 1gp4A Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1gp4A.1gp4AAKG370.if.pml - Pymol script for Figure 5 of service for comparative analysis of proteins.” Bioinformatics • 22:1656-7. 1gp4A.cbcvg - used by other 1gp4A – related pymol scripts 4.6 About report maker report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker

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