Pages 1–6 1cz4 Evolutionary trace report by report maker September 11, 2008 4.3.3 DSSP 5 4.3.4 HSSP 5 4.3.5 LaTex 5 4.3.6 Muscle 5 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 Protein Data Bank entry (PDB id 1cz4): Title: Nmr structure of vat-n: the n-terminal domain of vat (vcp- like atpase of thermoplasma) Compound: Mol id: 1; molecule: vcp-like atpase; chain: a; frag- ment: n-terminal domain: m1 to e183 followed by a diglycine spacer; engineered: yes Organism, scientific name: Thermoplasma Acidophilum; 1cz4 contains a single unique chain 1cz4A (185 residues long). This is an NMR-determined structure – in this report the first model in the file was used. CONTENTS 1 Introduction 1 2 CHAIN 1CZ4A 2.1 O05209 overview 2 Chain 1cz4A 1 2.1 O05209 overview 1 From SwissProt, id O05209, 100% identical to 1cz4A: Description: 2.2 Multiple sequence alignment for 1cz4A 1 VCP-like ATPase. Organism, scientific name: 2.3 Residue ranking in 1cz4A 1 Thermoplasma acidophilum. Taxonomy: 2.4 Top ranking residues in 1cz4A and their position on Archaea; Euryarchaeota; Thermoplasmata; Thermoplas- the structure 2 matales; Thermoplasmataceae; Thermoplasma. Biophysicochemical properties: 2.4.1 Clustering of residues at 25% coverage. 2 2.4.2 Possible novel functional surfaces at 25% Temperature dependence: Optimum temperature is 70 degrees coverage. 2 Celsius; Subunit: Homohexamer. Forms a ring-shaped particle. 3 Notes on using trace results 4 Similarity: Belongs to the AAA ATPase family. CDC48 subfamily. 3.1 Coverage 4 About: This Swiss-Prot entry is copyright. It is produced through a 3.2 Known substitutions 4 collaboration between the Swiss Institute of Bioinformatics and the 3.3 Surface 4 EMBL outstation - the European Bioinformatics Institute. There are 3.4 Number of contacts 4 no restrictions on its use as long as its content is in no way modified 3.5 Annotation 4 and this statement is not removed. 3.6 Mutation suggestions 5 2.2 Multiple sequence alignment for 1cz4A 4 Appendix 5 For the chain 1cz4A, the alignment 1cz4A.msf (attached) with 16 4.1 File formats 5 sequences was used. The alignment was assembled through combi- 4.2 Color schemes used 5 nation of BLAST searching on the UniProt database and alignment 4.3 Credits 5 using Muscle program. It can be found in the attachment to this 4.3.1 Alistat 5 report, under the name of 1cz4A.msf. Its statistics, from the alistat 4.3.2 CE 5 program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 1-185 in 1cz4A colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) Format: MSF Number of sequences: 16 Total number of residues: 2855 Smallest: 171 Largest: 185 Average length: 178.4 Alignment length: 185 Average identity: 44% Most related pair: 98% Fig. 2. Residues in 1cz4A, colored by their relative importance. Clockwise: Most unrelated pair: 31% front, back, top and bottom views. Most distant seq: 39% Furthermore, 9% of residues show as conserved in this alignment. The alignment consists of 12% prokaryotic, and 87% archaean sequences. The file containing the sequence descriptions can be found in the attachment, under the name 1cz4A.descr. 2.3 Residue ranking in 1cz4A The 1cz4A sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1cz4A can be found in the file called 1cz4A.ranks sorted in the attachment. 2.4 Top ranking residues in 1cz4A 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 1cz4A 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 Fig. 3. Residues in 1cz4A, colored according to the cluster they belong to: belong to. The clusters in Fig.3 are composed of the residues listed red, followed by blue and yellow are the largest clusters (see Appendix for in Table 1. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 1. cluster size member Table 1. continued color residues cluster size member red 21 9,11,13,14,18,19,20,22,43,44 color residues 46,52,66,70,71,74,76,78,82 blue 14 95,98,100,101,102,128,132 83,85 150,152,153,155,157,162,168 continued in next column yellow 3 177,179,181 continued in next column 2 Table 1. continued Table 2. continued cluster size member res type substitutions(%) cvg color residues 27 D P(6)D(87)T(6) 0.14 green 3 39,40,54 66 I V(6)I(87)E(6) 0.14 purple 2 25,68 82 G G(93)D(6) 0.15 azure 2 184,185 46 K G(87)K(12) 0.18 132 Q G(87)Q(12) 0.18 Table 1. Clusters of top ranking residues in 1cz4A. 13 E A(6)E(87)Q(6) 0.20 71 S G(81)S(12)R(6) 0.20 78 G E(6)K(6)G(87) 0.20 2.4.2 Possible novel functional surfaces at 25% coverage. One 153 V V(87)T(12) 0.21 group of residues is conserved on the 1cz4A surface, away from (or 83 D E(18)D(81) 0.22 susbtantially larger than) other functional sites and interfaces reco- 22 S G(81)K(6)S(12) 0.23 gnizable in PDB entry 1cz4. It is shown in Fig. 4. The residues 19 P S(12)V(75)P(12) 0.24 128 P P(75)V(25) 0.25 Table 2. Residues forming surface ”patch” in 1cz4A. Table 3. res type disruptive mutations 9 L (YR)(TH)(SKECG)(FQWD) 14 A (KYER)(QHD)(N)(FTMW) 25 R (TD)(SYEVCLAPIG)(FMW)(N) 68 R (TD)(SYEVCLAPIG)(FMW)(N) 70 D (R)(FWH)(KYVCAG)(TQM) 74 R (TD)(SYEVCLAPIG)(FMW)(N) 18 D (R)(FWH)(YVCAG)(K) 20 G (K)(ER)(QM)(D) 61 D (R)(FWH)(K)(Y) 76 N (Y)(FWH)(R)(TE) 27 D (R)(H)(FW)(K) 66 I (YR)(H)(T)(K) 82 G (R)(K)(FWH)(EQM) 46 K (Y)(FW)(T)(SVAHD) 132 Q (Y)(FWH)(T)(SVAD) 13 E (H)(FW)(Y)(R) Fig. 4. A possible active surface on the chain 1cz4A. 71 S (K)(FMWR)(H)(EQ) 78 G (FW)(HR)(Y)(KE) belonging to this surface ”patch” are listed in Table 2, while Table 153 V (KR)(E)(YQH)(D) 3 suggests possible disruptive replacements for these residues (see 83 D (R)(FWH)(YVCAG)(K) Section 3.6). 22 S (FWR)(H)(K)(YM) 19 P (R)(Y)(H)(K) Table 2. 128 P (YR)(H)(TKE)(SQCDG) res type substitutions(%) cvg 9 L L(100) 0.09 Table 3. Disruptive mutations for the surface patch in 1cz4A. 14 A A(100) 0.09 25 R R(100) 0.09 68 R R(100) 0.09 Another group of surface residues is shown in Fig.5. The right panel 70 D D(100) 0.09 shows (in blue) the rest of the larger cluster this surface belongs to. 74 R R(100) 0.09 The residues belonging to this surface ”patch” are listed in Table 18 D E(6)D(93) 0.10 4, while Table 5 suggests possible disruptive replacements for these 20 G G(93)Y(6) 0.12 residues (see Section 3.6). 61 D D(93)G(6) 0.12 Table 4. 76 N N(93)S(6) 0.12 res type substitutions(%) cvg continued in next column 101 A A(100) 0.09 continued in next column 3 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- sely, when looking for substitutions which will not affect the protein, 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 Fig. 5. Another possible active surface on the chain 1cz4A. The larger cluster To detect candidates for novel functional interfaces, first we look for residues that are solvent accessible (according to DSSP program) by it belongs to is shown in blue. 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 Table 4. continued that these residues form a “cluster” of residues which have neighbor res type substitutions(%) cvg within 5A˚ from any of their heavy atoms. 102 P P(100) 0.09 Note, however, that, if our picture of protein evolution is correct, 179 E E(93)D(6) 0.12 the neighboring residues which are not surface accessible might be 181 L L(93)I(6) 0.15 equally important in maintaining the interaction specificity - they 150 F L(12)F(87) 0.17 should not be automatically dropped from consideration when choo- 177 A E(6)V(68)A(25) 0.22 sing the set for mutagenesis.