Pages 1–6 1wp0 Evolutionary trace report by report maker August 11, 2010

4.3.1 Alistat 5 4.3.2 CE 5 4.3.3 DSSP 5 4.3.4 HSSP 5 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 6 4.7 Attachments 6

1 INTRODUCTION From the original Data Bank entry (PDB id 1wp0): Title: Human sco1 Compound: Mol id: 1; molecule: sco1 protein homolog; chain: a, b, c; fragment: ims fragment; synonym: hsco1; engineered: yes Organism, scientific name: Homo Sapiens; 1wp0 contains a single unique chain 1wp0A (160 residues long) and its homologues 1wp0C and 1wp0B. CONTENTS

1 Introduction 1 2 CHAIN 1WP0A 2.1 O75880 overview 2 Chain 1wp0A 1 2.1 O75880 overview 1 From SwissProt, id O75880, 98% identical to 1wp0A: 2.2 Multiple sequence alignment for 1wp0A 1 Description: SCO1 protein homolog, mitochondrial precursor. 2.3 Residue ranking in 1wp0A 1 Organism, scientific name: Homo sapiens (Human). 2.4 Top ranking residues in 1wp0A and their position on Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; the structure 1 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 2.4.1 Clustering of residues at 25% coverage. 2 Catarrhini; Hominidae; Homo. 2.4.2 Overlap with known functional surfaces at Function: Thought to play a role in either mitochondrial 25% coverage. 2 transport or insertion of copper into the active site of COX. 2.4.3 Possible novel functional surfaces at 25% Subcellular location: Mitochondrial. coverage. 3 Tissue specificity: Predominantly expressed in tissues characteri- zed by high rates of oxidative phosphorylation (OxPhos), including 3 Notes on using trace results 4 muscle, , and . 3.1 Coverage 4 Disease: Defects in SCO1 are a cause of 3.2 Known substitutions 4 deficiency (COX deficiency) [MIM:220110]. COX deficiency is a 3.3 Surface 4 clinically heterogeneous disorder. The clinical features are ranging 3.4 Number of contacts 4 from isolated to severe multisystem disease, with onset 3.5 Annotation 5 from infancy to adulthood. 3.6 suggestions 5 Similarity: Belongs to the SCO1/2 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 138-297 in 1wp0A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

2.2 Multiple sequence alignment for 1wp0A For the chain 1wp0A, the alignment 1wp0A.msf (attached) with 429 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 1wp0A.msf. Its statistics, from the alistat program are the following: Fig. 2. Residues in 1wp0A, colored by their relative importance. Clockwise: front, back, top and bottom views. Format: MSF Number of sequences: 429 Total number of residues: 64691 2.4.1 Clustering of residues at 25% coverage. Fig. 3 shows the Smallest: 94 top 25% of all residues, this time colored according to clusters they Largest: 160 belong to. The clusters in Fig.3 are composed of the residues listed Average length: 150.8 Alignment length: 160 Average identity: 34% Most related pair: 99% Most unrelated pair: 9% Most distant seq: 30%

Furthermore, <1% of residues show as conserved in this ali- gnment. The alignment consists of 8% eukaryotic ( 1% vertebrata, <1% arthropoda, 3% fungi, <1% plantae), and 11% prokaryotic 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 1wp0A.descr.

2.3 Residue ranking in 1wp0A The 1wp0A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1wp0A can be found in the file called 1wp0A.ranks sorted in the attachment.

2.4 Top ranking residues in 1wp0A and their position on the structure In the following we consider residues ranking among top 25% of Fig. 3. Residues in 1wp0A, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for residues in the protein . Figure 2 shows residues in 1wp0A colored the coloring scheme). Clockwise: front, back, top and bottom views. The by their importance: bright red and yellow indicate more conser- corresponding Pymol script is attached. ved/important residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. in Table 1.

2 Table 1. cluster size member color residues red 37 140,142,161,163,164,165,166 167,169,170,171,173,174,176 177,200,201,202,203,204,205 207,208,216,220,226,227,228 229,241,243,256,259,260,261 265,266

Table 1. Clusters of top ranking residues in 1wp0A.

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 1wp0B.Table 2 lists the top 25% of residues at the interface with 1wp0B. 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˚ ) 260 H H(97)G. 0.04 5/5 4.03 ASTK 261 T S(64) 0.15 33/8 3.08 T(30)M A(1).GQ NIDV 241 Y Y(51)V 0.17 3/3 4.11 F(36) L(6)N W(3)IRM 265 Y Y(73) 0.18 7/0 4.23 V(4) A(2) F(12) L(3).IM RTE 259 D D(72) 0.20 37/3 2.87 A(2) N(6)H T(3). E(4)R G(2) S(4)VMK L

Table 2. The top 25% of residues in 1wp0A at the interface with 1wp0B. (Field names: res: residue number in the PDB entry; type: 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. )

3 Table 3. res type disruptive 260 H (E)(MD)(Q)(K) 261 T (R)(K)(H)(FW) 241 Y (K)(E)(Q)(R) 265 Y (K)(Q)(R)(E) 259 D (R)(H)(FW)(Y)

Table 3. List of disruptive mutations for the top 25% of residues in 1wp0A, that are at the interface with 1wp0B.

Fig. 5. A possible active surface on the chain 1wp0A. The larger cluster it belongs to is shown in blue.

Table 4. continued res type substitutions(%) cvg 169 C C(98)S(1).P 0.04 260 H H(97)G.ASTK 0.04 170 P P(92)K(3)DA(1)V 0.05 RS.LE 164 F F(94)I(2)LV(1). 0.06 XY 207 R R(95)FN(2)HYKA. 0.06 CI 205 P P(96)AS(1)IF.GH 0.07 L 256 Y Y(92)VQ.(3)HRDT 0.07 GNLI 171 D D(84)E(7)TR(1) 0.09 N(1)A(1).YHSK 216 Y Y(84)F(12)W(1)R 0.09 MHA. 229 G G(86)A(4)SLP(5) 0.10 TVFM 167 T S(9)T(85)A(2)C 0.12 Fig. 4. Residues in 1wp0A, at the interface with 1wp0B, colored by their rela- L(1)G.VF tive importance. 1wp0B is shown in backbone representation (See Appendix 140 F F(84)L(4)W(3) 0.13 for the coloring scheme for the protein chain 1wp0A.) .(6)YIM 166 F F(63)Y(35)LC.V 0.14 Figure 4 shows residues in 1wp0A colored by their importance, at the 220 F F(84)Y(3)M(2) 0.14 interface with 1wp0B. L(2)V(3)SXI(1)N HWQAT 2.4.3 Possible novel functional surfaces at 25% coverage. One 261 T S(64)T(30)MA(1) 0.15 group of residues is conserved on the 1wp0A surface, away from (or .GQNIDV susbtantially larger than) other functional sites and interfaces reco- 241 Y Y(51)VF(36)L(6) 0.17 gnizable in PDB entry 1wp0. It is shown in Fig. 5. The right panel NW(3)IRM shows (in blue) the rest of the larger cluster this surface belongs to. 265 Y Y(73)V(4)A(2) 0.18 The residues belonging to this surface ”patch” are listed in Table F(12)L(3).IMRTE 4, while Table 5 suggests possible disruptive replacements for these 259 D D(72)A(2)N(6)H 0.20 residues (see Section 3.6). T(3).E(4)RG(2) Table 4. S(4)VMKL res type substitutions(%) cvg 228 T T(70)S(10)GR(9) 0.21 208 D D(99). 0.01 NCFV(3)ALI(1)KM 174 P P(98)LI.ETM 0.03 continued in next column 204 D D(99)Q.ST 0.03 continued in next column

4 Table 4. continued couple of residues should affect the protein somehow, with the exact res type substitutions(%) cvg effects to be determined experimentally. Y(1)D 176 E E(24)T(38)D(10) 0.23 3.2 Known substitutions N(1)S(14)IMA(6) One of the table columns is “substitutions” - other amino acid types Q(1).GVF seen at the same position in the alignment. These amino acid types 203 I C(9)V(56)I(18) 0.23 may be interchangeable at that position in the protein, so if one wants L(13)FAGMT. to affect the protein by a point mutation, they should be avoided. For 243 V V(66)I(10)T(6) 0.25 example if the substitutions are “RVK” and the original protein has F(2)A(8)SNL(2)R an R at that position, it is advisable to try anything, but RVK. Conver- PMYE.G 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 1wp0A. 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 3.3 Surface 208 D (R)(FWH)(VCAG)(KY) 174 P (R)(Y)(H)(T) To detect candidates for novel functional interfaces, first we look for 204 D (R)(FWH)(Y)(K) residues that are solvent accessible (according to DSSP program) by ˚ 2 169 C (R)(K)(E)(H) at least 10A , which is roughly the area needed for one water mole- 260 H (E)(MD)(Q)(K) cule to come in the contact with the residue. Furthermore, we require 170 P (Y)(R)(H)(T) that these residues form a “cluster” of residues which have neighbor ˚ 164 F (K)(E)(Q)(R) within 5A from any of their heavy atoms. 207 R (D)(T)(E)(Y) Note, however, that, if our picture of protein evolution is correct, 205 P (R)(Y)(TE)(K) the neighboring residues which are not surface accessible might be 256 Y (K)(QM)(R)(E) equally important in maintaining the interaction specificity - they 171 D (R)(FW)(H)(Y) should not be automatically dropped from consideration when choo- 216 Y (K)(Q)(E)(M) sing the set for mutagenesis. (Especially if they form a cluster with 229 G (R)(K)(E)(H) the surface residues.) 167 T (KR)(Q)(H)(E) 3.4 Number of contacts 140 F (K)(E)(T)(Q) 166 F (K)(E)(Q)(D) Another column worth noting is denoted “noc/bb”; it tells the num- 220 F (KE)(D)(T)(QR) ber of contacts heavy atoms of the residue in question make across 261 T (R)(K)(H)(FW) the interface, as well as how many of them are realized through the 241 Y (K)(E)(Q)(R) backbone atoms (if all or most contacts are through the backbone, 265 Y (K)(Q)(R)(E) mutation presumably won’t have strong impact). Two heavy atoms ˚ 259 D (R)(H)(FW)(Y) are considered to be “in contact” if their centers are closer than 5A. 228 T (R)(K)(H)(FW) 3.5 Annotation 176 E (H)(FWR)(Y)(CG) 203 I (R)(Y)(H)(K) If the residue annotation is available (either from the pdb file or 243 V (R)(K)(Y)(E) 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 Table 5. Disruptive mutations for the surface patch in 1wp0A. 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 Trace results are commonly expressed in terms of coverage: the resi- they are meant to be disruptive to the interaction of the protein due is important if its “coverage” is small - that is if it belongs to with its ligand. The attempt is made to complement the following some small top percentage of residues [100% is all of the residues properties: small [AV GSTC], medium [LPNQDEMIK], large in a chain], according to trace. The ET results are presented in the [WFYHR], hydrophobic [LPVAMWFI], polar [GTCY ]; posi- form of a table, usually limited to top 25% percent of residues (or tively [KHR], or negatively [DE] charged, aromatic [WFYH], to some nearby percentage), sorted by the strength of the presumed long aliphatic chain [EKRQM], OH-group possession [SDETY ], evolutionary pressure. (I.e., the smaller the coverage, the stronger the and NH2 group possession [NQRK]. The suggestions are listed pressure on the residue.) Starting from the top of that list, mutating a according to how different they appear to be from the original amino

5 DarkOliveGreen, CornflowerBlue, grey55, burlywood, LimeGreen, tan, DarkOrange, DeepPink, maroon, BlanchedAlmond. The colors used to distinguish the residues by the estimated evolutionary pressure they experience can be seen in Fig. 6. COVERAGE 4.3 Credits

V 4.3.1 Alistat alistat reads a multiple sequence alignment from the 100% 50% 30% 5% file and shows a number of simple statistics about it. These stati- stics include the format, the number of sequences, the total number of residues, the average and range of the sequence lengths, and the alignment length (e.g. including gap characters). Also shown are some percent identities. A percent pairwise alignment identity is defi- ned as (idents / MIN(len1, len2)) where idents is the number of V exact identities and len1, len2 are the unaligned lengths of the two RELATIVE IMPORTANCE 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 Fig. 6. Coloring scheme used to color residues by their relative importance. 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 acid, and they are grouped in round brackets if they appear equally by HHMI/Washington University School of Medicine, 1992-2001, disruptive. From left to right, each bracketed group of amino acid and freely distributed under the GNU General Public License. types resembles more strongly the original (i.e. is, presumably, less 4.3.2 CE To map ligand binding sites from different disruptive) These suggestions are tentative - they might prove disrup- source structures, report maker uses the CE program: tive to the fold rather than to the interaction. Many researcher will http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) choose, however, the straightforward alanine mutations, especially in ”Protein structure alignment by incremental combinatorial extension the beginning stages of their investigation. (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.3.3 DSSP In this work a residue is considered solvent accessi- 4 APPENDIX ble if the DSSP program finds it exposed to water by at least 10A˚ 2, 4.1 File formats which is roughly the area needed for one water molecule to come in Files with extension “ranks sorted” are the actual trace results. The the contact with the residue. DSSP is copyrighted by W. Kabsch, C. fields in the table in this file: Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version 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 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 • rho ET score - the smaller this value, the lesser variability of 4.3.5 LaTex The text for this report was processed using LATEX; this position across the branches of the tree (and, presumably, Leslie Lamport, “LaTeX: A Document Preparation System Addison- the greater the importance for the protein) Wesley,” Reading, Mass. (1986). • cvg coverage - percentage of the residues on the structure which 4.3.6 Muscle When making alignments “from scratch”, report have this rho or smaller maker uses Muscle alignment program: Edgar, Robert C. (2004), • gaps percentage of gaps in this column ”MUSCLE: multiple sequence alignment with high accuracy and 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 by cluster size: black is a single-residue cluster; clusters composed of 4.3.7 Pymol The figures in this report were produced using more than one residue colored according to this hierarchy (ordered Pymol. The scripts can be found in the attachment. Pymol by descending size): red, blue, yellow, green, purple, azure, tur- is an open-source application copyrighted by DeLano Scien- quoise, brown, coral, magenta, LightSalmon, SkyBlue, violet, gold, tific LLC (2005). For more information about Pymol see bisque, LightSlateBlue, orchid, RosyBrown, MediumAquamarine, http://pymol.sourceforge.net/. (Note for Windows

6 users: the attached package needs to be unzipped for Pymol to read 4.6 About report maker the scripts and launch the viewer.) report maker was written in 2006 by Ivana Mihalek. The 1D ran- king visualization program was written by Ivica Res.ˇ report maker 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/ • 1wp0A.complex.pdb - coordinates of 1wp0A with all of its interacting partners The viewer is self-unpacking and self-installing. Input files to be used • 1wp0A.etvx - ET viewer input file for 1wp0A with ETV (extension .etvx) can be found in the attachment to the • main report. 1wp0A.cluster report.summary - Cluster report summary for 1wp0A 4.5 Citing this work • 1wp0A.ranks - Ranks file in sequence order for 1wp0A The method used to rank residues and make predictions in this report • 1wp0A.clusters - Cluster descriptions for 1wp0A can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of • 1wp0A.msf - the multiple sequence alignment used for the chain Evolution-Entropy Hybrid Methods for Ranking of Protein Residues 1wp0A by Importance” J. Mol. Bio. 336: 1265-82. For the original version • 1wp0A.descr - description of sequences used in 1wp0A msf of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- tionary Trace Method Defines Binding Surfaces Common to Protein • 1wp0A.ranks sorted - full listing of residues and their ranking Families” J. Mol. Bio. 257: 342-358. for 1wp0A report maker itself is described in Mihalek I., I. Res and O. • 1wp0A.1wp0B.if.pml - Pymol script for Figure 4 Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type • 1wp0A.cbcvg - used by other 1wp0A – related pymol scripts of service for comparative analysis of .” Bioinformatics 22:1656-7.

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