Pages 1–5 2qsb Evolutionary trace report by report maker September 11, 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 2qsb): Title: Crystal structure of a protein from uncharacterized family upf0147 from thermoplasma acidophilum Compound: Mol id: 1; molecule: upf0147 protein ta0600; chain: a; engineered: yes Organism, scientific name: Thermoplasma Acidophilum Dsm 1728; CONTENTS 2qsb contains a single unique chain 2qsbA (85 residues long). 1 Introduction 1 2 Chain 2qsbA 1 2.1 Q9HKJ8 overview 1 2 CHAIN 2QSBA 2.2 Multiple sequence alignment for 2qsbA 1 2.1 Q9HKJ8 overview 2.3 Residue ranking in 2qsbA 1 From SwissProt, id Q9HKJ8, 96% identical to 2qsbA: 2.4 Top ranking residues in 2qsbA and their position on Description: Hypothetical UPF0147 protein Ta0600. the structure 1 Organism, scientific name: Thermoplasma acidophilum. 2.4.1 Clustering of residues at 25% coverage. 2 Taxonomy: Archaea; Euryarchaeota; Thermoplasmata; Thermoplas- 2.4.2 Possible novel functional surfaces at 25% matales; Thermoplasmataceae; Thermoplasma. coverage. 3 Similarity: Belongs to the UPF0147 family. About: This Swiss-Prot entry is copyright. It is produced through a 3 Notes on using trace results 3 collaboration between the Swiss Institute of Bioinformatics and the 3.1 Coverage 3 EMBL outstation - the European Bioinformatics Institute. There are 3.2 Known substitutions 3 no restrictions on its use as long as its content is in no way modified 3.3 Surface 3 and this statement is not removed. 3.4 Number of contacts 4 3.5 Annotation 4 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 2qsbA 4 Appendix 4 For the chain 2qsbA, the alignment 2qsbA.msf (attached) with 33 4.1 File formats 4 sequences was used. The alignment was downloaded from the HSSP 4.2 Color schemes used 4 database, and fragments shorter than 75% of the query as well as 4.3 Credits 4 duplicate sequences were removed. It can be found in the attachment 4.3.1 Alistat 4 to this report, under the name of 2qsbA.msf. Its statistics, from the 4.3.2 CE 4 alistat program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 2-86 in 2qsbA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) Format: MSF Number of sequences: 33 Total number of residues: 2671 Smallest: 71 Largest: 85 Average length: 80.9 Alignment length: 85 Average identity: 45% Most related pair: 99% Most unrelated pair: 25% Most distant seq: 48% Furthermore, 5% of residues show as conserved in this alignment. Fig. 2. Residues in 2qsbA, colored by their relative importance. Clockwise: The alignment consists of 3% prokaryotic, and 39% archaean front, back, top and bottom views. 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 2qsbA.descr. 2.3 Residue ranking in 2qsbA The 2qsbA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2qsbA can be found in the file called 2qsbA.ranks sorted in the attachment. 2.4 Top ranking residues in 2qsbA and their position on the structure In the following we consider residues ranking among top 25% of residues in the protein . Figure 2 shows residues in 2qsbA 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. 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. Fig. 3. Residues in 2qsbA, colored according to the cluster they belong to: cluster size member red, followed by blue and yellow are the largest clusters (see Appendix for color residues the coloring scheme). Clockwise: front, back, top and bottom views. The red 20 16,25,26,27,28,29,30,33,40 corresponding Pymol script is attached. 58,62,64,65,66,68,72,76,80 82,83 susbtantially larger than) other functional sites and interfaces reco- Table 1. Clusters of top ranking residues in 2qsbA. gnizable in PDB entry 2qsb. It is shown in Fig. 4. The right panel shows (in blue) the rest of the larger cluster this surface belongs to. The residues belonging to this surface ”patch” are listed in Table 2.4.2 Possible novel functional surfaces at 25% coverage. One 2, while Table 3 suggests possible disruptive replacements for these group of residues is conserved on the 2qsbA surface, away from (or residues (see Section 3.6). 2 Table 2. res type substitutions(%) cvg 26 P P(100) 0.06 66 N N(100) 0.06 72 R R(100) 0.06 83 E E(100) 0.06 64 D D(96)N(3) 0.08 68 P P(96)T(3) 0.08 28 N N(93)T(6) 0.09 27 K K(15)R(84) 0.12 76 Y Y(12)W(81)L(6) 0.14 65 P P(90)Q(6)S(3) 0.15 Fig. 4. A possible active surface on the chain 2qsbA. The larger cluster it 25 V V(72)I(21)T(6) 0.19 belongs to is shown in blue. 30 R R(96)K(3) 0.20 80 S S(87)A(6)G(3) 0.22 T(3) 62 A A(12)S(75)L(3) 0.25 V(3)T(6) Table 2. Residues forming surface ”patch” in 2qsbA. Table 3. res type disruptive mutations 26 P (YR)(TH)(SKECG)(FQWD) 66 N (Y)(FTWH)(SEVCARG)(MD) 72 R (TD)(SYEVCLAPIG)(FMW)(N) 83 E (FWH)(YVCARG)(T)(SNKLPI) 64 D (R)(FWH)(Y)(VCAG) 68 P (R)(YH)(K)(E) 28 N (FYWH)(R)(E)(TVMA) 27 K (Y)(T)(FW)(SVCAG) 76 Y (K)(Q)(R)(E) 65 P (Y)(R)(H)(T) 25 V (R)(K)(Y)(E) 30 R (T)(YD)(SVCAG)(FELWPI) 80 S (KR)(QH)(FMW)(E) 62 A (R)(K)(YE)(H) Table 3. Disruptive mutations for the surface patch in 2qsbA. 3 NOTES ON USING TRACE RESULTS 3.1 Coverage Trace results are commonly expressed in terms of coverage: the resi- due is important if its “coverage” is small - that is if it belongs to some small top percentage of residues [100% is all of the residues in a chain], according to trace. The ET results are presented in the form of a table, usually limited to top 25% percent of residues (or to some nearby percentage), sorted by the strength of the presumed evolutionary pressure. (I.e., the smaller the coverage, the stronger the pressure on the residue.) Starting from the top of that list, mutating a couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types 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 to affect the protein by a point mutation, they should be avoided. For COVERAGE 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- V sely, when looking for substitutions which will not affect the protein, 100% 50% 30% 5% 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 V to 100%. RELATIVE IMPORTANCE 3.3 Surface To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by Fig. 5. Coloring scheme used to color residues by their relative importance. 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 disruptive) These suggestions are tentative - they might prove disrup- within 5A˚ from any of their heavy atoms. tive to the fold rather than to the interaction. Many researcher will Note, however, that, if our picture of protein evolution is correct, choose, however, the straightforward alanine mutations, especially in the neighboring residues which are not surface accessible might be the beginning stages of their investigation. equally important in maintaining the interaction specificity - they should not be automatically dropped from consideration when choo- 4 APPENDIX sing the set for mutagenesis. (Especially if they form a cluster with 4.1 File formats the surface residues.) Files with extension “ranks sorted” are the actual trace results. The 3.4 Number of contacts fields in the table in this file: Another column worth noting is denoted “noc/bb”; it tells the num- • alignment# number of the position in the alignment 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 • residue# residue number in the PDB file backbone atoms (if all or most contacts are through the backbone, • type amino acid type mutation presumably won’t have strong impact).