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3A1j Lichtarge Lab 2006 Pages 1–17 3a1j Evolutionary trace report by report maker June 8, 2009 4 Chain 3a1jB 11 4.1 O60921 overview 11 4.2 Multiple sequence alignment for 3a1jB 11 4.3 Residue ranking in 3a1jB 11 4.4 Top ranking residues in 3a1jB and their position on the structure 11 4.4.1 Clustering of residues at 25% coverage. 11 4.4.2 Overlap with known functional surfaces at 25% coverage. 12 4.4.3 Possible novel functional surfaces at 25% coverage. 13 5 Notes on using trace results 15 5.1 Coverage 15 5.2 Known substitutions 15 5.3 Surface 15 5.4 Number of contacts 15 5.5 Annotation 15 5.6 Mutation suggestions 15 6 Appendix 15 6.1 File formats 15 CONTENTS 6.2 Color schemes used 16 6.3 Credits 16 1 Introduction 1 6.3.1 Alistat 16 6.3.2 CE 16 2 Chain 3a1jA 1 6.3.3 DSSP 16 2.1 Q99638 overview 1 6.3.4 HSSP 16 2.2 Multiple sequence alignment for 3a1jA 1 6.3.5 LaTex 16 2.3 Residue ranking in 3a1jA 1 6.3.6 Muscle 16 2.4 Top ranking residues in 3a1jA and their position on 6.3.7 Pymol 16 the structure 1 6.4 Note about ET Viewer 16 2.4.1 Clustering of residues at 25% coverage. 2 6.5 Citing this work 16 2.4.2 Overlap with known functional surfaces at 6.6 About report maker 17 25% coverage. 2 6.7 Attachments 17 2.4.3 Possible novel functional surfaces at 25% coverage. 5 1 INTRODUCTION From the original Protein Data Bank entry (PDB id 3a1j): 3 Chain 3a1jC 6 Title: Crystal structure of the human rad9-hus1-rad1 complex 3.1 Q5R7X9 overview 6 Compound: Mol id: 1; molecule: cell cycle checkpoint control 3.2 Multiple sequence alignment for 3a1jC 6 protein rad9a; chain: a; fragment: n-terminal domain, residues 1- 3.3 Residue ranking in 3a1jC 7 266; synonym: hrad9, dna repair exonuclease rad9 homolog a; ec: 3.4 Top ranking residues in 3a1jC and their position on 3.1.11.2; engineered: yes; mol id: 2; molecule: checkpoint pro- the structure 7 tein hus1; chain: b; synonym: hhus1; engineered: yes; mol id: 3; 3.4.1 Clustering of residues at 25% coverage. 7 molecule: cell cycle checkpoint protein rad1; chain: c; fragment: resi- 3.4.2 Overlap with known functional surfaces at dues 13-275; synonym: hrad1, dna repair exonuclease rad1 homolog, 25% coverage. 8 rad1- like dna damage checkpoint protein; ec: 3.1.11.2; engineered: 3.4.3 Possible novel functional surfaces at 25% yes coverage. 9 Organism, scientific name: Homo Sapiens; 1 Lichtarge lab 2006 Fig. 1. Residues 1-132 in 3a1jA colored by their relative importance. (See Fig. 2. Residues 133-266 in 3a1jA colored by their relative importance. (See Appendix, Fig.25, for the coloring scheme.) Appendix, Fig.25, for the coloring scheme.) 3a1j contains unique chains 3a1jA (265 residues), 3a1jC (263 2.4 Top ranking residues in 3a1jA and their position on residues), and 3a1jB (269 residues) the structure In the following we consider residues ranking among top 25% of resi- dues in the protein . Figure 3 shows residues in 3a1jA colored by their 2 CHAIN 3A1JA importance: bright red and yellow indicate more conserved/important 2.1 Q99638 overview residues (see Appendix for the coloring scheme). A Pymol script for producing this figure can be found in the attachment. From SwissProt, id Q99638, 93% identical to 3a1jA: Description: Cell cycle checkpoint control protein (RAD9 homolog A) (S. pombe). Organism, scientific name: Homo sapiens (Human). Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Catarrhini; Hominidae; Homo. 2.2 Multiple sequence alignment for 3a1jA For the chain 3a1jA, the alignment 3a1jA.msf (attached) with 10 sequences was used. The alignment was assembled through combi- nation of BLAST searching on the UniProt database and alignment using Muscle program. It can be found in the attachment to this report, under the name of 3a1jA.msf. Its statistics, from the alistat program are the following: Format: MSF Number of sequences: 10 Total number of residues: 2599 Smallest: 248 Largest: 265 Average length: 259.9 Alignment length: 265 Average identity: 30% Most related pair: 81% Fig. 3. Residues in 3a1jA, colored by their relative importance. Clockwise: Most unrelated pair: 18% front, back, top and bottom views. Most distant seq: 25% 2.4.1 Clustering of residues at 25% coverage. Fig. 4 shows the Furthermore, 3% of residues show as conserved in this alignment. top 25% of all residues, this time colored according to clusters they The alignment consists of 90% eukaryotic ( 30% vertebrata, 20% belong to. The clusters in Fig.4 are composed of the residues listed arthropoda, 20% fungi) sequences. (Descriptions of some sequences in Table 1. were not readily available.) The file containing the sequence descrip- tions can be found in the attachment, under the name 3a1jA.descr. Table 1. cluster size member 2.3 Residue ranking in 3a1jA color residues red 51 12,15,16,20,21,22,23,24,26 The 3a1jA sequence is shown in Figs. 1–2, with each residue colored 30,36,39,41,42,43,46,47,53 according to its estimated importance. The full listing of residues in 3a1jA can be found in the file called 3a1jA.ranks sorted in the continued in next column attachment. 2 Table 2. continued res type subst’s cvg noc/ dist (%) bb (A˚ ) 196 E T(29) 0.16 35/30 2.81 E(59) Q(10) Table 2. The top 25% of residues in 3a1jA at the interface with 3a1jB. (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 number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 3. res type disruptive mutations 159 F (T)(KE)(DR)(QCG) 195 T (KR)(FQMWH)(NELPI)(D) 196 E (FWH)(Y)(R)(VA) Fig. 4. Residues in 3a1jA, colored according to the cluster they belong to: Table 3. List of disruptive mutations for the top 25% of residues in 3a1jA, red, followed by blue and yellow are the largest clusters (see Appendix for that are at the interface with 3a1jB. the coloring scheme). Clockwise: front, back, top and bottom views. The corresponding Pymol script is attached. Table 1. continued cluster size member color residues 57,58,61,78,81,84,132,150 159,164,165,180,195,196,216 217,218,220,221,222,223,224 228,229,230,244,246,260,261 262,263,264,265 blue 9 7,95,97,112,114,115,117,120 121 Table 1. Clusters of top ranking residues in 3a1jA. 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 3a1jB.Table 2 lists the top 25% of residues at the interface with 3a1jB. The following table (Table 3) suggests possible disruptive replacements for these residues (see Section 5.6). Table 2. Fig. 5. Residues in 3a1jA, at the interface with 3a1jB, colored by their rela- res type subst’s cvg noc/ dist tive importance. 3a1jB is shown in backbone representation (See Appendix (%) bb (A˚ ) for the coloring scheme for the protein chain 3a1jA.) 159 F F(80) 0.07 5/5 3.16 L(10) M(10) Figure 5 shows residues in 3a1jA colored by their importance, at the 195 T T(69) 0.16 30/10 3.12 interface with 3a1jB. S(29) Sucrose binding site. Table 4 lists the top 25% of residues at the continued in next column interface with 3a1jASUC6001 (sucrose). The following table (Table 5) suggests possible disruptive replacements for these residues (see Section 5.6). 3 Table 4. res type subst’s cvg noc/ dist antn (%) bb (A˚ ) 132 L L(59) 0.21 1/1 4.33 I(20) Q(10) M(10) 39 R T(29) 0.23 16/0 3.64 site K(20) R(50) Table 4. The top 25% of residues in 3a1jA at the interface with sucrose.(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 number of contacts realized through backbone atoms given in the bracket; dist: distance of closest apporach to the ligand. ) Table 5. res type disruptive mutations 132 L (Y)(R)(TH)(SCG) 39 R (D)(TY)(FEVLAWPI)(CG) Table 5. List of disruptive mutations for the top 25% of residues in 3a1jA, that are at the interface with sucrose. Fig. 6. Residues in 3a1jA, at the interface with sucrose, colored by their rela- tive importance. The ligand (sucrose) is colored green. Atoms further than 30A˚ away from the geometric center of the ligand, as well as on the line of sight to the ligand were removed. (See Appendix for the coloring scheme for the protein chain 3a1jA.) 4 Figure 6 shows residues in 3a1jA colored by their importance, at the interface with 3a1jASUC6001. Interface with 3a1jC.Table 6 lists the top 25% of residues at the interface with 3a1jC. The following table (Table 7) suggests possible disruptive replacements for these residues (see Section 5.6). Table 6.
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