Pages 1–6 1pft Evolutionary trace report by report maker August 6, 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 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 6 4.7 Attachments 6 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 1pft): Title: N-terminal domain of tfiib, nmr Compound: Mol id: 1; molecule: tfiib; chain: a; fragment: n- terminal domain; synonym: pftfiibn; engineered: yes Organism, scientific name: Pyrococcus Furiosus; 1pft contains a single unique chain 1pftA (50 residues long). This is an NMR-determined structure – in this report the first model in the file was used. CONTENTS 2 CHAIN 1PFTA 1 Introduction 1 2.1 P61999 overview 2 Chain 1pftA 1 From SwissProt, id P61999, 97% identical to 1pftA: 2.1 P61999 overview 1 Description: Transcription initiation factor IIB (TFIIB). 2.2 Multiple sequence alignment for 1pftA 1 Organism, scientific name: Pyrococcus woesei. 2.3 Residue ranking in 1pftA 1 Taxonomy: Archaea; Euryarchaeota; Thermococci; Thermococca- 2.4 Top ranking residues in 1pftA and their position on les; Thermococcaceae; Pyrococcus. the structure 1 Function: Stabilizes TBP binding to an archaeal box-A promo- 2.4.1 Clustering of residues at 26% coverage. 2 ter. Also responsible for recruiting RNA polymerase II to the pre- 2.4.2 Overlap with known functional surfaces at initiation complex (DNA-TBP-TFIIB). 26% coverage. 3 Cofactor: Binds 1 zinc ion per subunit. 2.4.3 Possible novel functional surfaces at 26% Similarity: Belongs to the TFIIB family. coverage. 3 Similarity: Contains 1 TFIIB-type zinc finger. 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 3.5 Annotation 4 2.2 Multiple sequence alignment for 1pftA 3.6 Mutation suggestions 4 For the chain 1pftA, the alignment 1pftA.msf (attached) with 134 sequences was used. The alignment was downloaded from the HSSP 4 Appendix 4 database, and fragments shorter than 75% of the query as well as 4.1 File formats 4 duplicate sequences were removed. It can be found in the attachment 4.2 Color schemes used 5 to this report, under the name of 1pftA.msf. Its statistics, from the 4.3 Credits 5 alistat program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 1-50 in 1pftA colored by their relative importance. (See Appendix, Fig.6, for the coloring scheme.) Format: MSF Number of sequences: 134 Total number of residues: 6148 Smallest: 40 Largest: 50 Average length: 45.9 Alignment length: 50 Average identity: 46% Most related pair: 98% Most unrelated pair: 20% Most distant seq: 45% Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 5% eukaryotic ( <1% vertebrata, 1% arthropoda, 1% fungi, <1% plantae), 9% prokaryotic, and 32% archaean sequences. (Descriptions of some sequences were not rea- Fig. 2. Residues in 1pftA, colored by their relative importance. Clockwise: dily available.) The file containing the sequence descriptions can be front, back, top and bottom views. found in the attachment, under the name 1pftA.descr. 2.3 Residue ranking in 1pftA The 1pftA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 1pftA can be found in the file called 1pftA.ranks sorted in the attachment. 2.4 Top ranking residues in 1pftA and their position on the structure In the following we consider residues ranking among top 26% of resi- dues in the protein (the closest this analysis allows us to get to 25%). Figure 2 shows residues in 1pftA 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 26% coverage. Fig. 3 shows the top 26% 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 color residues Fig. 3. Residues in 1pftA, colored according to the cluster they belong to: red 7 8,11,24,27,30,31,33 red, followed by blue and yellow are the largest clusters (see Appendix for blue 5 42,43,44,45,46 the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 1. Clusters of top ranking residues in 1pftA. 3) suggests possible disruptive replacements for these residues (see 2.4.2 Overlap with known functional surfaces at 26% coverage. Section 3.6). The name of the ligand is composed of the source PDB identifier and the heteroatom name used in that file. Zinc ion binding site. Table 2 lists the top 26% of residues at the interface with 1pftZN51 (zinc ion). The following table (Table 2 Table 2. res type subst’s cvg noc/ dist (%) bb (A˚ ) 27 C C(100) 0.02 6/1 2.35 30 C C(94) 0.06 9/3 2.33 T(5)D 11 C C(96) 0.08 9/3 2.33 H(3) 31 G G(97) 0.10 1/0 4.75 A(2) 8 C C(94)A 0.20 5/1 2.34 S(1)D .(1)G Table 2. The top 26% of residues in 1pftA at the interface with zinc ion.(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 num- ber of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 3. Fig. 4. Residues in 1pftA, at the interface with zinc ion, colored by their res type disruptive relative importance. The ligand (zinc ion) is colored green. Atoms further mutations than 30A˚ away from the geometric center of the ligand, as well as on the line 27 C (KER)(FQMWHD)(NYLPI)(SVA) of sight to the ligand were removed. (See Appendix for the coloring scheme 30 C (R)(K)(FWH)(E) for the protein chain 1pftA.) 11 C (E)(K)(QMDR)(FNLWPI) 31 G (KER)(QHD)(FYMW)(N) 8 C (R)(K)(E)(H) Table 3. List of disruptive mutations for the top 26% of residues in 1pftA, that are at the interface with zinc ion. Figure 4 shows residues in 1pftA colored by their importance, at the interface with 1pftZN51. 2.4.3 Possible novel functional surfaces at 26% coverage. One group of residues is conserved on the 1pftA surface, away from (or susbtantially larger than) other functional sites and interfaces reco- gnizable in PDB entry 1pft. It is shown in Fig. 5. The residues belonging to this surface ”patch” are listed in Table 4, while Table 5 suggests possible disruptive replacements for these residues (see Section 3.6). Table 4. res type substitutions(%) cvg 27 C C(100) 0.02 30 C C(94)T(5)D 0.06 11 C C(96)H(3) 0.08 31 G G(97)A(2) 0.10 Fig. 5. A possible active surface on the chain 1pftA. 33 V V(96)I(3) 0.12 24 E E(80)Q(3)D(10)Y 0.24 IMNT(2) Table 5. res type disruptive Table 4. Residues forming surface ”patch” in 1pftA. mutations continued in next column 3 Table 5. continued the interface, as well as how many of them are realized through the res type disruptive backbone atoms (if all or most contacts are through the backbone, mutations mutation presumably won’t have strong impact). Two heavy atoms 27 C (KER)(FQMWHD)(NYLPI)(SVA) are considered to be “in contact” if their centers are closer than 5A˚ . 30 C (R)(K)(FWH)(E) 11 C (E)(K)(QMDR)(FNLWPI) 3.5 Annotation 31 G (KER)(QHD)(FYMW)(N) If the residue annotation is available (either from the pdb file or 33 V (YR)(KE)(H)(QD) from other sources), another column, with the header “annotation” 24 E (H)(FW)(R)(Y) 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 1pftA. 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.