Pages 1–6 1q8r Evolutionary trace report by report maker December 23, 2009

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 6 4.5 Citing this work 6 4.6 About report maker 6 4.7 Attachments 6

1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1q8r): Title: Structure of e.coli rusa holliday junction resolvase Compound: Mol id: 1; molecule: crossover junction endodeoxyribo- nuclease rusa; chain: a, b; synonym: rusa resolvase; holliday junction nuclease rusa; holliday juction resolvase; endodeoxyribonuclease rus; ec: 3.1.22.-; engineered: yes Organism, scientific name: Escherichia Coli; CONTENTS 1q8r contains a single unique chain 1q8rA (118 residues long) and 1 Introduction 1 its homologue 1q8rB.

2 Chain 1q8rA 1 2.1 P40116 overview 1 2 CHAIN 1Q8RA 2.2 Multiple sequence alignment for 1q8rA 1 2.1 P40116 overview 2.3 Residue ranking in 1q8rA 1 2.4 Top ranking residues in 1q8rA and their position on From SwissProt, id P40116, 100% identical to 1q8rA: the structure 2 Description: Crossover junction endodeoxyribonuclease rusA (EC 2.4.1 Clustering of residues at 25% coverage. 2 3.1.22.-) (Holliday junction nuclease rusA) (Holliday juction resol- 2.4.2 Overlap with known functional surfaces at vase). 25% coverage. 2 Organism, scientific name: Escherichia coli, and Escherichia coli 2.4.3 Possible novel functional surfaces at 25% O6. coverage. 3 Taxonomy: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Escherichia. 3 Notes on using trace results 4 Function: Endonuclease that resolves holliday junction intermedia- 3.1 Coverage 4 tes in genetic recombination. Cleaves X-junctions by introducing 3.2 Known substitutions 4 symmetrical nicks in two strands of the same polarity. Processes Hol- 3.3 Surface 4 liday intermediates made by recA. Corrects the defects in genetic 3.4 Number of contacts 4 recombination and DNA repair associated with inactivation of ruvAB 3.5 Annotation 4 or ruvC. 3.6 Mutation suggestions 4 Similarity: Belongs to the rusA family. About: This Swiss-Prot entry is copyright. It is produced through a 4 Appendix 5 collaboration between the Swiss Institute of Bioinformatics and the 4.1 File formats 5 EMBL outstation - the European Bioinformatics Institute. There are 4.2 Color schemes used 5 no restrictions on its use as long as its content is in no way modified 4.3 Credits 5 and this statement is not removed.

1 Lichtarge lab 2006 Fig. 1. Residues 1-118 in 1q8rA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

2.2 Multiple sequence alignment for 1q8rA For the chain 1q8rA, the alignment 1q8rA.msf (attached) with 59 sequences was used. The alignment was downloaded from the HSSP database, and fragments shorter than 75% of the query as well as duplicate sequences were removed. It can be found in the attachment to this report, under the name of 1q8rA.msf. Its statistics, from the alistat program are the following:

Format: MSF Number of sequences: 59 Total number of residues: 6556 Fig. 2. Residues in 1q8rA, colored by their relative importance. Clockwise: front, back, top and bottom views. Smallest: 48 Largest: 118 Average length: 111.1 Alignment length: 118 Average identity: 44% Most related pair: 99% Most unrelated pair: 10% Most distant seq: 33%

Furthermore, 1% of residues show as conserved in this alignment. The alignment consists of 16% prokaryotic, and 10% viral 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 1q8rA.descr. 2.3 Residue ranking in 1q8rA The 1q8rA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1q8rA can be found in the file called 1q8rA.ranks sorted in the attachment. 2.4 Top ranking residues in 1q8rA and their position on the structure

In the following we consider residues ranking among top 25% of resi- Fig. 3. Residues in 1q8rA, colored according to the cluster they belong to: dues in the protein . Figure 2 shows residues in 1q8rA colored by their red, followed by blue and yellow are the largest clusters (see Appendix for importance: bright red and yellow indicate more conserved/important the coloring scheme). Clockwise: front, back, top and bottom views. The residues (see Appendix for the coloring scheme). A Pymol script for corresponding Pymol script is attached. producing this figure can be found in the attachment.

2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Table 1. top 25% of all residues, this time colored according to clusters they cluster size member belong to. The clusters in Fig.3 are composed of the residues listed color residues in Table 1. continued in next column

2 Table 1. continued cluster size member color residues red 29 8,9,11,12,13,14,15,17,18,28 31,64,65,69,70,71,72,73,74 76,77,79,80,81,82,85,86,90 91

Table 1. Clusters of top ranking residues in 1q8rA.

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. Interface with 1q8rB.Table 2 lists the top 25% of residues at the interface with 1q8rB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 3.6).

Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 73 N N(100) 0.02 6/2 3.59 76 K K(100) 0.02 6/0 3.96 Fig. 4. Residues in 1q8rA, at the interface with 1q8rB, colored by their rela- 72 D D(93) 0.03 59/23 2.93 tive importance. 1q8rB is shown in backbone representation (See Appendix N(1) for the coloring scheme for the protein chain 1q8rA.) A(5) 70 D D(98) 0.05 1/0 4.97 .(1) susbtantially larger than) other functional sites and interfaces reco- 71 L L(79) 0.14 29/8 3.49 gnizable in PDB entry 1q8r. It is shown in Fig. 5. The right panel P(8) shows (in blue) the rest of the larger cluster this surface belongs to. I(5) V(5) M(1)

Table 2. The top 25% of residues in 1q8rA at the interface with 1q8rB. (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 number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. )

Table 3. res type disruptive Fig. 5. A possible active surface on the chain 1q8rA. The larger cluster it mutations belongs to is shown in blue. 73 N (Y)(FTWH)(SEVCARG)(MD) 76 K (Y)(FTW)(SVCAG)(HD) The residues belonging to this surface ”patch” are listed in Table 72 D (R)(H)(FW)(Y) 4, while Table 5 suggests possible disruptive replacements for these 70 D (R)(FWH)(VCAG)(KY) residues (see Section 3.6). 71 L (Y)(R)(H)(T) Table 4. Table 3. List of disruptive mutations for the top 25% of residues in res type substitutions(%) cvg 1q8rA, that are at the interface with 1q8rB. 73 N N(100) 0.02 76 K K(100) 0.02 72 D D(93)N(1)A(5) 0.03 Figure 4 shows residues in 1q8rA colored by their importance, at the 80 D D(98)G(1) 0.03 interface with 1q8rB. continued in next column 2.4.3 Possible novel functional surfaces at 25% coverage. One group of residues is conserved on the 1q8rA surface, away from (or

3 Table 4. continued Table 5. continued res type substitutions(%) cvg res type disruptive 90 D D(94)N(3).(1) 0.04 mutations 70 D D(98).(1) 0.05 15 N (Y)(FWH)(T)(VCARG) 11 P P(93).(6) 0.07 8 L (R)(Y)(H)(T) 15 N N(93)D(3).(3) 0.08 69 R (T)(D)(E)(CG) 8 L L(89).(8)S(1) 0.09 91 D (FWR)(H)(YVA)(KCG) 69 R R(79)S(3)I(6) 0.10 9 P (Y)(R)(T)(H) K(1)W(5)F(1) 12 P (YR)(H)(T)(KE) .(1) 71 L (Y)(R)(H)(T) 91 D D(93)R(3)G(1) 0.11 18 Y (K)(Q)(EM)(NR) .(1) 14 N (Y)(H)(T)(R) 9 P P(89).(8)L(1) 0.12 13 S (KR)(FWH)(QM)(LPI) 12 P P(86).(6)V(3) 0.13 85 A (R)(K)(E)(Y) I(3) 64 P (R)(Y)(H)(T) 71 L L(79)P(8)I(5) 0.14 65 D (R)(H)(FW)(K) V(5)M(1) 86 G (R)(E)(K)(FWHD) 18 Y W(74)H(3)Y(16) 0.16 79 F (K)(E)(R)(TQD) L(1).(3) 74 L (R)(Y)(T)(E) 14 N V(67)N(16)A(6) 0.17 28 S (FW)(R)(H)(K) .(3)L(5) 108 G (R)(K)(E)(H) 13 S T(35)S(61).(3) 0.18 85 A A(76)S(8)V(10) 0.19 Table 5. Disruptive mutations for the surface patch in 1q8rA. C(1)I(1)G(1) 64 P P(86)A(3)L(1) 0.20 D(1)T(1).(3) N(1) 3 NOTES ON USING TRACE RESULTS 65 D D(76)T(8)N(5) 0.20 3.1 Coverage S(3)A(3).(3) Trace results are commonly expressed in terms of coverage: the resi- 86 G G(84)K(3)A(5) 0.21 due is important if its “coverage” is small - that is if it belongs to V(1).(1)H(1) some small top percentage of residues [100% is all of the residues E(1) in a chain], according to trace. The ET results are presented in the 79 F L(54)F(32)M(5) 0.22 form of a table, usually limited to top 25% percent of residues (or I(1)S(3)C(3) to some nearby percentage), sorted by the strength of the presumed 74 L I(45)L(27)A(6) 0.23 evolutionary pressure. (I.e., the smaller the coverage, the stronger the F(1)Y(11)R(6) pressure on the residue.) Starting from the top of that list, mutating a 28 S S(84)R(5).(3) 0.24 couple of residues should affect the protein somehow, with the exact K(1)T(1)N(1) effects to be determined experimentally. C(1) 108 G G(81)Y(3)P(1) 0.25 3.2 Known substitutions R(3).(3)E(3) One of the table columns is “substitutions” - other amino acid types A(3) seen at the same position in the alignment. These amino acid types may be interchangeable at that position in the protein, so if one wants Table 4. Residues forming surface ”patch” in 1q8rA. to affect the protein by a point mutation, they should be avoided. For 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 5. sely, when looking for substitutions which will not affect the protein, res type disruptive one may try replacing, R with K, or (perhaps more surprisingly), with mutations V. The percentage of times the substitution appears in the alignment 73 N (Y)(FTWH)(SEVCARG)(MD) is given in the immediately following bracket. No percentage is given 76 K (Y)(FTW)(SVCAG)(HD) in the cases when it is smaller than 1%. This is meant to be a rough 72 D (R)(H)(FW)(Y) guide - due to rounding errors these percentages often do not add up 80 D (R)(FWH)(K)(Y) to 100%. 90 D (R)(FWH)(YVCAG)(T) 70 D (R)(FWH)(VCAG)(KY) 3.3 Surface 11 P (YR)(TH)(SCG)(KE) To detect candidates for novel functional interfaces, first we look for continued in next column 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

4 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 COVERAGE should not be automatically dropped from consideration when choo-

sing the set for mutagenesis. (Especially if they form a cluster with V the surface residues.) 100% 50% 30% 5% 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, V mutation presumably won’t have strong impact). Two heavy atoms RELATIVE IMPORTANCE are considered to be “in contact” if their centers are closer than 5A˚ .

3.5 Annotation Fig. 6. Coloring scheme used to color residues by their relative importance. 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 • rho ET score - the smaller this value, the lesser variability of (indicating existence of related site record in PDB ), S-S (disulfide this position across the branches of the tree (and, presumably, bond forming residue), hb (hydrogen bond forming residue, jb (james the greater the importance for the protein) bond forming residue), and sb (for salt bridge forming residue). • cvg coverage - percentage of the residues on the structure which 3.6 Mutation suggestions have this rho or smaller • gaps Mutation suggestions are completely heuristic and based on comple- percentage of gaps in this column mentarity with the substitutions found in the alignment. Note that 4.2 Color schemes used they are meant to be disruptive to the interaction of the protein with its ligand. The attempt is made to complement the following The following color scheme is used in figures with residues colored properties: small [AV GSTC], medium [LPNQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- more than one residue colored according to this hierarchy (ordered 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. 6. disruptive) These suggestions are tentative - they might prove disrup- 4.3 Credits tive to the fold rather than to the interaction. Many researcher will 4.3.1 Alistat alistat reads a multiple sequence alignment from the choose, however, the straightforward alanine mutations, especially in file and shows a number of simple statistics about it. These stati- the beginning stages of their investigation. stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the 4 APPENDIX 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 seq” is calculated by finding the maximum pairwise identity (best • type amino acid type 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:

5 http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) The viewer is self-unpacking and self-installing. Input files to be used ”Protein structure alignment by incremental combinatorial extension with ETV (extension .etvx) can be found in the attachment to the (CE) of the optimal path . Protein Engineering 11(9) 739-747. main report. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4.5 Citing this work ˚ 2 ble if the DSSP program finds it exposed to water by at least 10A , The method used to rank residues and make predictions in this report which is roughly the area needed for one water molecule to come in can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of the contact with the residue. DSSP is copyrighted by W. Kabsch, C. Evolution-Entropy Hybrid Methods for Ranking of Protein Residues Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version by Importance” J. Mol. Bio. 336: 1265-82. For the original version @cmbi.kun.nl by Elmar.Krieger November 18,2002, of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- http://www.cmbi.kun.nl/gv/dssp/descrip.html. tionary Trace Method Defines Binding Surfaces Common to Protein Families” J. Mol. Bio. 257: 342-358. 4.3.4 HSSP Whenever available, report maker uses HSSP ali- report maker itself is described in Mihalek I., I. Res and O. gnment as a starting point for the analysis (sequences shorter than Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 75% of the query are taken out, however); R. Schneider, A. de of service for comparative analysis of proteins.” Bioinformatics Daruvar, and C. Sander. ”The HSSP database of protein structure- 22:1656-7. sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. 4.6 About report maker http://swift.cmbi.kun.nl/swift/hssp/ report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 4.3.5 LaTex The text for this report was processed using LATEX; is copyrighted by Lichtarge Lab, Baylor College of Medicine, Leslie Lamport, “LaTeX: A Document Preparation System Addison- Houston. Wesley,” Reading, Mass. (1986). 4.7 Attachments 4.3.6 Muscle When making alignments “from scratch”, report maker uses Muscle alignment program: Edgar, Robert C. (2004), The following files should accompany this report: ”MUSCLE: multiple sequence alignment with high accuracy and • 1q8rA.complex.pdb - coordinates of 1q8rA with all of its inter- high throughput.” Nucleic Acids Research 32(5), 1792-97. acting partners http://www.drive5.com/muscle/ • 1q8rA.etvx - ET viewer input file for 1q8rA • 4.3.7 Pymol The figures in this report were produced using 1q8rA.cluster report.summary - Cluster report summary for Pymol. The scripts can be found in the attachment. Pymol 1q8rA is an open-source application copyrighted by DeLano Scien- • 1q8rA.ranks - Ranks file in sequence order for 1q8rA tific LLC (2005). For more information about Pymol see • 1q8rA.clusters - Cluster descriptions for 1q8rA http://pymol.sourceforge.net/. (Note for Windows • 1q8rA.msf - the multiple sequence alignment used for the chain users: the attached package needs to be unzipped for Pymol to read 1q8rA the scripts and launch the viewer.) • 1q8rA.descr - description of sequences used in 1q8rA msf 4.4 Note about ET Viewer • 1q8rA.ranks sorted - full listing of residues and their ranking for Dan Morgan from the Lichtarge lab has developed a visualization 1q8rA tool specifically for viewing trace results. If you are interested, please • 1q8rA.1q8rB.if.pml - Pymol script for Figure 4 visit: • 1q8rA.cbcvg - used by other 1q8rA – related pymol scripts http://mammoth.bcm.tmc.edu/traceview/

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