Pages 1–6 1ls9 Evolutionary trace report by report maker September 26, 2008

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 1ls9): Title: Structure of the cytochrome c6 from the green alga glomerata Compound: Mol id: 1; molecule: cytochrome c6; chain: a Organism, scientific name: Cladophora Glomerata 1ls9 contains a single unique chain 1ls9A (91 residues long).

2 CHAIN 1LS9A CONTENTS 2.1 P83391 overview 1 Introduction 1 From SwissProt, id P83391, 100% identical to 1ls9A: Description: Cytochrome c6 (Soluble cytochrome f) (Cytochrome 2 Chain 1ls9A 1 c553) (Cytochrome c- 553). 2.1 P83391 overview 1 Organism, scientific name: Cladophora glomerata. 2.2 Multiple sequence alignment for 1ls9A 1 : Eukaryota; ; ; ; 2.3 Residue ranking in 1ls9A 1 ; Cladophoraceae; Cladophora. 2.4 Top ranking residues in 1ls9A and their position on Function: Functions as an electron carrier between membrane- the structure 1 bound cytochrome b6f and photosystem I in oxygenic photosynthesis 2.4.1 Clustering of residues at 25% coverage. 2 (By similarity). 2.4.2 Overlap with known functional surfaces at Subunit: Monomer. 25% coverage. 2 Subcellular location: Chloroplast; within the thylakoid lumen. 2.4.3 Possible novel functional surfaces at 25% Ptm: Binds 1 heme group per subunit. coverage. 3 Similarity: Belongs to the cytochrome c family. PetJ subfamily. About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 4 collaboration between the Swiss Institute of Bioinformatics and the 3.1 Coverage 4 EMBL outstation - the European Bioinformatics Institute. There are 3.2 Known substitutions 4 no restrictions on its use as long as its content is in no way modified 3.3 Surface 4 and this statement is not removed. 3.4 Number of contacts 4 2.2 Multiple sequence alignment for 1ls9A 3.5 Annotation 4 3.6 Mutation suggestions 4 For the chain 1ls9A, the alignment 1ls9A.msf (attached) with 28 sequences was used. The alignment was assembled through combi- 4 Appendix 5 nation of BLAST searching on the UniProt database and alignment 4.1 File formats 5 using Muscle program. It can be found in the attachment to this 4.2 Color schemes used 5 report, under the name of 1ls9A.msf. Its statistics, from the alistat 4.3 Credits 5 program are the following:

1 Lichtarge lab 2006 Fig. 1. Residues 1-91 in 1ls9A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.)

Format: MSF Number of sequences: 28 Total number of residues: 2459 Smallest: 81 Largest: 91 Average length: 87.8 Alignment length: 91 Average identity: 54% Most related pair: 99% Most unrelated pair: 35% Most distant seq: 56%

Furthermore, 9% of residues show as conserved in this alignment. Fig. 2. Residues in 1ls9A, colored by their relative importance. Clockwise: The alignment consists of 46% eukaryotic ( 14% plantae), and front, back, top and bottom views. 50% 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 1ls9A.descr. 2.3 Residue ranking in 1ls9A The 1ls9A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1ls9A can be found in the file called 1ls9A.ranks sorted in the attachment. 2.4 Top ranking residues in 1ls9A and their position on the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 2 shows residues in 1ls9A colored by their importance: bright red and yellow indicate more conserved/important 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. 3 shows the top 25% of all residues, this time colored according to clusters they belong to. The clusters in Fig.3 are composed of the residues listed in Table 1. Table 1. cluster size member Fig. 3. Residues in 1ls9A, colored according to the cluster they belong to: color residues red, followed by blue and yellow are the largest clusters (see Appendix for red 23 9,13,16,17,18,20,21,24,26,33 the coloring scheme). Clockwise: front, back, top and bottom views. The 34,55,56,59,62,63,64,70,75 corresponding Pymol script is attached. 78,81,82,91

HEM binding site. Table 2 lists the top 25% of residues at the Table 1. Clusters of top ranking residues in 1ls9A. interface with 1ls9AHEM92 (hem). The following table (Table 3) suggests possible disruptive replacements for these residues (see 2.4.2 Overlap with known functional surfaces at 25% coverage. Section 3.6). The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file.

2 Table 2. Table 3. continued res type subst’s cvg noc/ dist res type disruptive (%) bb (A˚ ) mutations 13 F F(100) 0.10 2/0 4.66 70 L (YR)(TH)(SKECG)(FQWD) 17 C C(100) 0.10 39/21 1.81 33 T (R)(K)(H)(FW) 34 L L(100) 0.10 20/3 3.41 62 A (Y)(R)(E)(H) 63 M M(100) 0.10 91/23 2.28 82 V (YR)(KE)(H)(QD) 78 V V(100) 0.10 7/1 3.91 26 N (FWH)(Y)(R)(E) 20 C C(96) 0.13 27/8 1.91 56 V (YR)(KE)(H)(QD) A(3) 55 Q (Y)(H)(T)(FW) 21 H H(96) 0.13 79/0 1.99 T(3) Table 3. List of disruptive mutations for the top 25% of residues in 1ls9A, 16 N N(92) 0.15 15/13 3.44 that are at the interface with HEM. H(7) 64 P P(92) 0.15 22/4 3.64 S(7) 70 L L(96) 0.18 1/0 4.25 I(3) 33 T T(85) 0.20 14/14 2.62 N(7) V(7) 62 A A(85) 0.20 18/17 3.81 N(7) P(7) 82 V V(96) 0.21 3/0 3.88 I(3) 26 N N(92) 0.22 22/0 3.19 T(3) G(3) 56 V V(92) 0.23 10/0 3.61 L(3) I(3) 55 Q Q(92) 0.24 25/0 3.31 L(3) I(3)

Table 2. The top 25% of residues in 1ls9A at the interface with HEM.(Field names: res: residue number in the PDB entry; type: amino acid type; substs: substitutions seen in the alignment; with the percentage of each Fig. 4. Residues in 1ls9A, at the interface with HEM, colored by their relative A type in the bracket; noc/bb: number of contacts with the ligand, with the num- importance. The ligand (HEM) is colored green. Atoms further than 30 ˚ ber of contacts realized through backbone atoms given in the bracket; dist: away from the geometric center of the ligand, as well as on the line of sight distance of closest apporach to the ligand. ) to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 1ls9A.)

Table 3. res type disruptive Figure 4 shows residues in 1ls9A colored by their importance, at the mutations interface with 1ls9AHEM92. 13 F (KE)(TQD)(SNCRG)(M) 2.4.3 Possible novel functional surfaces at 25% coverage. One 17 C (KER)(FQMWHD)(NYLPI)(SVA) group of residues is conserved on the 1ls9A surface, away from (or 34 L (YR)(TH)(SKECG)(FQWD) susbtantially larger than) other functional sites and interfaces reco- 63 M (Y)(TH)(SCRG)(FWD) gnizable in PDB entry 1ls9. It is shown in Fig. 5. The residues 78 V (KYER)(QHD)(N)(FTMW) belonging to this surface ”patch” are listed in Table 4, while Table 20 C (KER)(QHD)(FYMW)(N) 5 suggests possible disruptive replacements for these residues (see 21 H (E)(QM)(KD)(NLPI) Section 3.6). 16 N (TY)(E)(SFVCAWG)(MHDR) 64 P (R)(Y)(H)(K) continued in next column

3 Table 5. continued res type disruptive mutations 34 L (YR)(TH)(SKECG)(FQWD) 59 G (KER)(FQMWHD)(NYLPI)(SVA) 63 M (Y)(TH)(SCRG)(FWD) 78 V (KYER)(QHD)(N)(FTMW) 81 Y (K)(QM)(NEVLAPIR)(D) 91 W (KE)(TQD)(SNCRG)(M) 20 C (KER)(QHD)(FYMW)(N) 21 H (E)(QM)(KD)(NLPI) 16 N (TY)(E)(SFVCAWG)(MHDR) 64 P (R)(Y)(H)(K) 70 L (YR)(TH)(SKECG)(FQWD) 75 I (Y)(R)(TH)(SCG) 33 T (R)(K)(H)(FW) 62 A (Y)(R)(E)(H) 82 V (YR)(KE)(H)(QD) 26 N (FWH)(Y)(R)(E) 56 V (YR)(KE)(H)(QD) 55 Q (Y)(H)(T)(FW) 18 A (R)(KY)(E)(H) Fig. 5. A possible active surface on the chain 1ls9A. Table 5. Disruptive mutations for the surface patch in 1ls9A. Table 4. res type substitutions(%) cvg 13 F F(100) 0.10 3 NOTES ON USING TRACE RESULTS 17 C C(100) 0.10 34 L L(100) 0.10 3.1 Coverage 59 G G(100) 0.10 Trace results are commonly expressed in terms of coverage: the resi- 63 M M(100) 0.10 due is important if its “coverage” is small - that is if it belongs to 78 V V(100) 0.10 some small top percentage of residues [100% is all of the residues 81 Y Y(100) 0.10 in a chain], according to trace. The ET results are presented in the 91 W W(100) 0.10 form of a table, usually limited to top 25% percent of residues (or 20 C C(96)A(3) 0.13 to some nearby percentage), sorted by the strength of the presumed 21 H H(96)T(3) 0.13 evolutionary pressure. (I.e., the smaller the coverage, the stronger the 16 N N(92)H(7) 0.15 pressure on the residue.) Starting from the top of that list, mutating a 64 P P(92)S(7) 0.15 couple of residues should affect the protein somehow, with the exact 70 L L(96)I(3) 0.18 effects to be determined experimentally. 75 I I(96)M(3) 0.18 33 T T(85)N(7)V(7) 0.20 3.2 Known substitutions 62 A A(85)N(7)P(7) 0.20 One of the table columns is “substitutions” - other amino acid types 82 V V(96)I(3) 0.21 seen at the same position in the alignment. These amino acid types 26 N N(92)T(3)G(3) 0.22 may be interchangeable at that position in the protein, so if one wants 56 V V(92)L(3)I(3) 0.23 to affect the protein by a point mutation, they should be avoided. For 55 Q Q(92)L(3)I(3) 0.24 example if the substitutions are “RVK” and the original protein has 18 A A(85)S(10)P(3) 0.25 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, Table 4. Residues forming surface ”patch” in 1ls9A. 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 Table 5. in the cases when it is smaller than 1%. This is meant to be a rough res type disruptive guide - due to rounding errors these percentages often do not add up mutations to 100%. 13 F (KE)(TQD)(SNCRG)(M) 17 C (KER)(FQMWHD)(NYLPI)(SVA) 3.3 Surface continued in next column To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by

4 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, COVERAGE the neighboring residues which are not surface accessible might be equally important in maintaining the interaction specificity - they V should not be automatically dropped from consideration when choo- 100% 50% 30% 5% 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 V the interface, as well as how many of them are realized through the RELATIVE IMPORTANCE 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˚ . Fig. 6. Coloring scheme used to color residues by their relative importance. 3.5 Annotation If the residue annotation is available (either from the pdb file or from other sources), another column, with the header “annotation” 2. their type 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 they are meant to be disruptive to the interaction of the protein 4.2 Color schemes used 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 GST C], medium [LP NQDEMIK], large by cluster size: black is a single-residue cluster; clusters composed of [W F Y HR], hydrophobic [LP V AMW F I], polar [GT CY ]; posi- more than one residue colored according to this hierarchy (ordered tively [KHR], or negatively [DE] charged, aromatic [W F Y H], by descending size): red, blue, yellow, green, purple, azure, tur- long aliphatic chain [EKRQM], OH-group possession [SDET Y ], 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- 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 4.1 File formats alignment length (e.g. including gap characters). Also shown are Files with extension “ranks sorted” are the actual trace results. The some percent identities. A percent pairwise alignment identity is defi- fields in the table in this file: ned as (idents / MIN(len1, len2)) where idents is the number of exact identities and len1, len2 are the unaligned lengths of the two • alignment# number of the position in the alignment sequences. The ”average percent identity”, ”most related pair”, and • residue# residue number in the PDB file ”most unrelated pair” of the alignment are the average, maximum, 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 • rank rank of the position according to older version of ET relative) for all N sequences, then finding the minimum of these N • variability has two subfields: numbers (hence, the most outlying sequence). alistat is copyrighted 1. number of different amino acids appearing in in this column by HHMI/Washington University School of Medicine, 1992-2001, of the alignment and freely distributed under the GNU General Public License.

5 4.3.2 CE To map ligand binding sites from different The viewer is self-unpacking and self-installing. Input files to be used source structures, report maker uses the CE program: with ETV (extension .etvx) can be found in the attachment to the http://cl.sdsc.edu/. Shindyalov IN, Bourne PE (1998) main report. ”Protein structure alignment by incremental combinatorial extension (CE) of the optimal path . Protein Engineering 11(9) 739-747. 4.5 Citing this work The method used to rank residues and make predictions in this report 4.3.3 DSSP In this work a residue is considered solvent accessi- 2 can be found in Mihalek, I., I. Res,ˇ O. Lichtarge. (2004). ”A Family of ble if the DSSP program finds it exposed to water by at least 10A˚ , Evolution-Entropy Hybrid Methods for Ranking of Protein Residues which is roughly the area needed for one water molecule to come in by Importance” J. Mol. Bio. 336: 1265-82. For the original version the contact with the residue. DSSP is copyrighted by W. Kabsch, C. of ET see O. Lichtarge, H.Bourne and F. Cohen (1996). ”An Evolu- Sander and MPI-MF, 1983, 1985, 1988, 1994 1995, CMBI version tionary Trace Method Defines Binding Surfaces Common to Protein by [email protected] November 18,2002, Families” J. Mol. Bio. 257: 342-358. http://www.cmbi.kun.nl/gv/dssp/descrip.html. report maker itself is described in Mihalek I., I. Res and O. Lichtarge (2006). ”Evolutionary Trace Report Maker: a new type 4.3.4 HSSP Whenever available, report maker uses HSSP ali- of service for comparative analysis of proteins.” Bioinformatics gnment as a starting point for the analysis (sequences shorter than 22:1656-7. 75% of the query are taken out, however); R. Schneider, A. de Daruvar, and C. Sander. ”The HSSP database of protein structure- 4.6 About report maker sequence alignments.” Nucleic Acids Res., 25:226–230, 1997. report maker was written in 2006 by Ivana Mihalek. The 1D ran- http://swift.cmbi.kun.nl/swift/hssp/ king visualization program was written by Ivica Res.ˇ report maker is copyrighted by Lichtarge Lab, Baylor College of Medicine, 4.3.5 LaTex The text for this report was processed using LAT X; E Houston. Leslie Lamport, “LaTeX: A Document Preparation System Addison- Wesley,” Reading, Mass. (1986). 4.7 Attachments 4.3.6 Muscle When making alignments “from scratch”, report The following files should accompany this report: maker uses Muscle alignment program: Edgar, Robert C. (2004), ”MUSCLE: multiple sequence alignment with high accuracy and • 1ls9A.complex.pdb - coordinates of 1ls9A with all of its inter- high throughput.” Nucleic Acids Research 32(5), 1792-97. acting partners http://www.drive5.com/muscle/ • 1ls9A.etvx - ET viewer input file for 1ls9A • 1ls9A.cluster report.summary - Cluster report summary for 4.3.7 Pymol The figures in this report were produced using 1ls9A Pymol. The scripts can be found in the attachment. Pymol • is an open-source application copyrighted by DeLano Scien- 1ls9A.ranks - Ranks file in sequence order for 1ls9A tific LLC (2005). For more information about Pymol see • 1ls9A.clusters - Cluster descriptions for 1ls9A http://pymol.sourceforge.net/. (Note for Windows • 1ls9A.msf - the multiple sequence alignment used for the chain users: the attached package needs to be unzipped for Pymol to read 1ls9A the scripts and launch the viewer.) • 1ls9A.descr - description of sequences used in 1ls9A msf 4.4 Note about ET Viewer • 1ls9A.ranks sorted - full listing of residues and their ranking for Dan Morgan from the Lichtarge lab has developed a visualization 1ls9A tool specifically for viewing trace results. If you are interested, please • 1ls9A.1ls9AHEM92.if.pml - Pymol script for Figure 4 visit: • 1ls9A.cbcvg - used by other 1ls9A – related pymol scripts http://mammoth.bcm.tmc.edu/traceview/

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