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2C6u Lichtarge Lab 2006

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2C6u Lichtarge Lab 2006 Pages 1–5 2c6u Evolutionary trace report by report maker June 29, 2010 4.3.3 DSSP 4 4.3.4 HSSP 4 4.3.5 LaTex 4 4.3.6 Muscle 4 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 2c6u): Title: Crystal structure of human clec-2 (clec1b) Compound: Mol id: 1; molecule: clec1b protein; chain: a; fragment: c-type lectin-like domain, residues 100-221; synonym: clec-2, c-type CONTENTS lectin-like receptor-2; engineered: yes Organism, scientific name: Homo Sapiens; 1 Introduction 1 2c6u contains a single unique chain 2c6uA (122 residues long). 2 Chain 2c6uA 1 2.1 Q6UWX7 overview 1 2.2 Multiple sequence alignment for 2c6uA 1 2.3 Residue ranking in 2c6uA 1 2.4 Top ranking residues in 2c6uA and their position on 2 CHAIN 2C6UA the structure 1 2.4.1 Clustering of residues at 25% coverage. 1 2.1 Q6UWX7 overview 2.4.2 Possible novel functional surfaces at 25% From SwissProt, id Q6UWX7, 100% identical to 2c6uA: coverage. 2 Description: QDED721. Organism, scientific name: Homo sapiens (Human). 3 Notes on using trace results 3 Taxonomy: Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; 3.1 Coverage 3 Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; 3.2 Known substitutions 3 Catarrhini; Hominidae; Homo. 3.3 Surface 3 3.4 Number of contacts 3 3.5 Annotation 3 3.6 Mutation suggestions 4 2.2 Multiple sequence alignment for 2c6uA 4 Appendix 4 For the chain 2c6uA, the alignment 2c6uA.msf (attached) with 53 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 2c6uA.msf. Its statistics, from the 4.3.2 CE 4 alistat program are the following: 1 Lichtarge lab 2006 Fig. 1. Residues 100-221 in 2c6uA colored by their relative importance. (See Appendix, Fig.5, for the coloring scheme.) Format: MSF Number of sequences: 53 Total number of residues: 6229 Smallest: 97 Largest: 122 Average length: 117.5 Alignment length: 122 Average identity: 32% Most related pair: 98% Most unrelated pair: 13% Most distant seq: 31% Fig. 2. Residues in 2c6uA, colored by their relative importance. Clockwise: front, back, top and bottom views. Furthermore, 2% of residues show as conserved in this alignment. The alignment consists of 41% eukaryotic ( 41% vertebrata) 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 2c6uA.descr. 2.3 Residue ranking in 2c6uA The 2c6uA sequence is shown in Fig. 1, with each residue colored according to its estimated importance. The full listing of residues in 2c6uA can be found in the file called 2c6uA.ranks sorted in the attachment. 2.4 Top ranking residues in 2c6uA 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 2c6uA 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. Fig. 3. Residues in 2c6uA, colored according to the cluster they belong to: red, followed by blue and yellow are the largest clusters (see Appendix for Table 1. the coloring scheme). Clockwise: front, back, top and bottom views. The cluster size member corresponding Pymol script is attached. color residues red 30 102,103,106,112,113,114,116 123,126,130,134,135,137,138 Table 1. continued 139,140,142,145,158,160,161 cluster size member 170,172,174,175,176,195,208 color residues continued in next column 216,217 Table 1. Clusters of top ranking residues in 2c6uA. 2 2.4.2 Possible novel functional surfaces at 25% coverage. One Table 2. continued group of residues is conserved on the 2c6uA surface, away from (or res type substitutions(%) cvg antn susbtantially larger than) other functional sites and interfaces reco- L(3)G(3) gnizable in PDB entry 2c6u. It is shown in Fig. 4. The right panel 135 A S(39)A(56)L(3) 0.21 shows (in blue) the rest of the larger cluster this surface belongs to. 112 S N(33)H(1)S(52) 0.22 R(1)K(5)F(1) Y(1) 116 F F(71)T(3)V(3) 0.23 Y(3)I(13)L(3) 145 I D(16)E(71)T(5) 0.24 I(1)A(1)V(1) 142 N S(60)N(22)D(11) 0.25 T(3)K(1) Table 2. Residues forming surface ”patch” in 2c6uA. Table 3. Fig. 4. A possible active surface on the chain 2c6uA. The larger cluster it res type disruptive belongs to is shown in blue. mutations 113 C (KER)(FQMWHD)(NYLPI)(SVA) The residues belonging to this surface ”patch” are listed in Table 208 C (KER)(FQMWHD)(NLPI)(Y) 2, while Table 3 suggests possible disruptive replacements for these 114 Y (K)(QR)(EM)(NVA) residues (see Section 3.6). 106 W (E)(K)(TD)(Q) 123 W (K)(E)(Q)(D) Table 2. 174 D (R)(FWH)(Y)(VCAG) res type substitutions(%) cvg antn 137 L (Y)(T)(R)(ECHG) 113 C C(100) 0.03 S-S 102 C (KER)(FQMWHD)(NLPI)(Y) 208 C C(98).(1) 0.04 S-S 170 W (K)(E)(Q)(D) 114 Y Y(98)L(1) 0.05 172 W (E)(K)(D)(Q) 106 W W(96).(1)F(1) 0.07 175 G (R)(FEWH)(K)(Y) 123 W W(96)Y(1)F(1) 0.07 139 K (Y)(FW)(T)(H) 174 D D(90)N(9) 0.08 195 C (KER)(H)(D)(Q) 137 L L(92)M(5)H(1) 0.10 134 N (Y)(FW)(H)(R) 102 C C(90).(9) 0.11 S-S 217 E (FWH)(Y)(CG)(VA) 170 W W(92)F(1)G(1) 0.12 103 D (R)(H)(FKW)(Y) Y(1)S(1) 176 S (R)(K)(H)(FQW) 172 W W(90)S(1)R(1) 0.12 135 A (R)(KY)(E)(H) T(3)N(1) 112 S (K)(M)(R)(E) 175 G G(88)Q(1)S(3) 0.13 116 F (K)(E)(QR)(D) N(5) 145 I (R)(Y)(H)(K) 139 K K(58)V(15)T(1) 0.16 142 N (Y)(FW)(H)(R) S(15)I(3)Q(5) 195 C C(83)S(3)A(3) 0.16 S-S Table 3. M(1)I(3)Y(1) Disruptive mutations for the surface patch in 2c6uA. R(1) 134 N N(54)G(26)S(5) 0.17 Y(1)D(7)K(3) 217 E M(15)Q(9)E(54) 0.19 3 NOTES ON USING TRACE RESULTS .(3)K(16) 3.1 Coverage 103 D P(73)D(11)L(3) 0.20 A(1).(1)H(3) Trace results are commonly expressed in terms of coverage: the resi- S(1)T(1) due is important if its “coverage” is small - that is if it belongs to 176 S S(60)T(30)I(1) 0.20 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 continued in next column 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 3 couple of residues should affect the protein somehow, with the exact effects to be determined experimentally. 3.2 Known substitutions One of the table columns is “substitutions” - other amino acid types COVERAGE 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 V to affect the protein by a point mutation, they should be avoided. For 100% 50% 30% 5% 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 V in the cases when it is smaller than 1%. This is meant to be a rough RELATIVE IMPORTANCE guide - due to rounding errors these percentages often do not add up to 100%. Fig. 5. Coloring scheme used to color residues by their 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 2 acid, and they are grouped in round brackets if they appear equally at least 10A˚ , which is roughly the area needed for one water mole- disruptive.
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